RuvbL1 and RuvbL2 enhance aggresome formation and...

Article

RuvbL1 and RuvbL2 enhance aggresome formationand disaggregate amyloid fibrilsNava Zaarur1, Xiaobin Xu2, Patrick Lestienne3, Anatoli B Meriin1, Mark McComb2,

Catherine E Costello1,2, Gary P Newnam4, Rakhee Ganti4, Nina V Romanova5,

Maruda Shanmugasundaram6, Sara TN Silva7, Tiago M Bandeiras8, Pedro M Matias7,8,

Kirill S Lobachev4, Igor K Lednev6, Yury O Chernoff4,5,* & Michael Y Sherman1,**

Abstract

The aggresome is an organelle that recruits aggregated proteinsfor storage and degradation. We performed an siRNA screen forproteins involved in aggresome formation and identified novelmammalian AAA+ protein disaggregases RuvbL1 and RuvbL2.Depletion of RuvbL1 or RuvbL2 suppressed aggresome formationand caused buildup of multiple cytoplasmic aggregates. Similarly,downregulation of RuvbL orthologs in yeast suppressed theformation of an aggresome-like body and enhanced the aggregatetoxicity. In contrast, their overproduction enhanced the resistanceto proteotoxic stress independently of chaperone Hsp104.Mammalian RuvbL associated with the aggresome, and theaggresome substrate synphilin-1 interacted directly with theRuvbL1 barrel-like structure near the opening of the central chan-nel. Importantly, polypeptides with unfolded structures andamyloid fibrils stimulated the ATPase activity of RuvbL. Finally,disassembly of protein aggregates was promoted by RuvbL. Thesedata indicate that RuvbL complexes serve as chaperones in proteindisaggregation.

Keywords aggresome; amyloid; disaggregation; RuvbL

Subject Categories Protein Biosynthesis & Quality Control

DOI 10.15252/embj.201591245 | Received 10 February 2015 | Revised 9 July

2015 | Accepted 13 July 2015

Introduction

Molecular chaperones and the ubiquitin-proteasome system (UPS)

promote refolding and degradation of abnormal polypeptides.

However, in aging and disease, these systems fail to repair or

destroy abnormal polypeptides, which then tend to form small cyto-

plasmic aggregates that cause cell toxicity, leading to various

protein misfolding disorders (Sherman & Goldberg, 2001; Meriin &

Sherman, 2005). A special cellular machinery has evolved to trans-

port such aggregates to the centrosome, forming an organelle called

the aggresome (Johnston et al, 1998; Chung et al, 2001; Webb et al,

2004; Corboy et al, 2005). The aggresome serves as a storage

compartment for protein aggregates and may be actively involved in

their refolding and proteasomal or autophagic degradation. It has

been proposed that the aggresome represents a protective cellular

response to the buildup of aggregating abnormal polypeptides that

occurs when chaperones and the UPS fail to handle abnormal

species (Tanaka et al, 2004; Olzmann et al, 2008), for example

during aging or disease.

A number of factors have been implicated in aggresome forma-

tion, including a microtubule-associated histone deacetylase,

HDAC6, PLIC, ataxin 3, Hsp70, or Bag3 (Kawaguchi et al, 2003;

Burnett & Pittman, 2005; Heir et al, 2006; Marx et al, 2007;

Gamerdinger et al, 2011; Zhang & Qian, 2011). Despite these findings,

the basic molecular mechanisms of aggresome formation are poorly

understood.

Here, we developed an unbiased, full-genome siRNA screen for

factors involved in aggresome formation in mammalian cells and

identified more than 100 knockdowns that significantly affect aggre-

some formation. We have focused on a pair of homologous proteins,

RuvbL1 and RuvbL2. These proteins belong to the AAA+ (adenosine

triphosphatases associated with diverse cellular activities) super-

family of ATPases. AAA+ proteins usually form hexameric or dode-

cameric ring structures and are characterized by the presence of the

AAA+ module, which contains the highly conserved Walker A and

1 Department of Biochemistry, Boston University School of Medicine, Boston, MA, USA2 Center for Biomedical Mass Spectrometry, Boston University School of Medicine, Boston, MA, USA3 INSERM U 1053, University of Bordeaux Segalen, Bordeaux, France4 School of Biology, Georgia Institute of Technology, Atlanta, GA, USA5 Laboratory of Amyloid Biology and Institute of Translational Biomedicine, St. Petersburg State University, St. Petersburg, Russia6 Department of Chemistry, University at Albany, State University of New York, Albany, NY, USA7 Instituto de Tecnologia Química e Biológica António Xavier, Universidade Nova de Lisboa, Oeiras, Portugal8 Instituto de Biologia Experimental e Tecnológica, Oeiras, Portugal

*Corresponding author. Tel: +1 404 894 1157; E-mail: [email protected]**Corresponding author. Tel: +1 617 638 5971; E-mail: [email protected]

ª 2015 The Authors The EMBO Journal 1

Published online: August 24, 2015

Walker B motifs responsible for nucleotide binding and hydrolysis,

respectively (Walker et al, 1982). Some AAA+ proteins, for exam-

ple, Hsp104 or ClpB (Doyle & Wickner, 2009; Winkler et al, 2012;

Clare & Saibil, 2013), serve as major molecular chaperones which

can promote disaggregation of protein aggregates, frequently work-

ing together with Hsp70 and Hsp40. Yeast Hsp104 is also involved

in formation of quality control protein deposits (Erjavec et al, 2007)

and controls the fragmentation and propagation of endogenous

amyloids, termed yeast prions (Chernoff et al, 1995). The genes

coding for the chaperones of the Hsp104/ClpB family, while present

in all kingdoms including bacteria, plants, various protists, and

fungi, are absent from the nuclear genomes of Metazoa. Though it

was demonstrated that the chaperone system Hsp70-Hsp40-Hsp110

can promote protein disaggregation in mammalian cells (Bukau

et al, 2006; Winkler et al, 2012; Rampelt et al, 2012; Mattoo &

Goloubinoff, 2013; Torrente & Shorter, 2014), the contribution of this

triad to the overall protein disaggregation has not been defined, and

an AAA+ disaggregase in mammalian cells may have been missed.

RuvbL1 and RuvbL2 proteins share sequence similarity to the

bacterial RuvB helicase (~30%) (Tsaneva et al, 1993; Putnam et al,

2001; Yamada et al, 2001). This similarity suggested that mamma-

lian RuvbL may also be a DNA helicase. In line with this notion,

RuvbL1 was found to be associated with the human replication

protein (RP)A3 (Qiu et al, 1998). Furthermore, DNA helicase activ-

ity of RuvbL complex was demonstrated in in vitro experiments

upon deletion of its auto-inhibitory domain II (Gorynia et al, 2011).

However, a number of publications suggest that RuvbL proteins

may have additional functions unrelated to helicase, since they are

involved in multiple protein complexes, for example, those includ-

ing TATA-binding protein (TBP) (Kanemaki et al, 1997), the large

RNA polymerase II holoenzyme (Qiu et al, 1998), chromatin remod-

eling factors (Shen et al, 2000; Jonsson et al, 2001; Jin et al, 2005;

Bakshi et al, 2006; Choi et al, 2009), certain transcription factors

(Jonsson et al, 2001; Ohdate et al, 2003), telomerase (Venteicher

et al, 2008), or phosphatidylinositol 3-kinase-related protein kinases

(Izumi et al, 2010). Furthermore, several studies have demonstrated

a role for RuvbL proteins in assembly of complexes, suggesting that

they may have chaperone-like activity (Machado-Pinilla et al,

2013). Here, we demonstrate that RuvbL functions as a general

molecular chaperone in protein quality control and facilitate

disaggregation of protein aggregates and amyloids.

Results

siRNA screening for genes involved in aggresome formation

To understand the molecular mechanisms underlying multiple steps

in the process of aggresome formation, we developed a high content

cell-based whole-genome siRNA screen (Fig 1A). We utilized two

reporters for aggresomes, RFP-fused ubiquitin (RFP-Ub), labeling

endogenous ubiquitinated proteins, and synphilin-1 tagged

with GFP (Syn-GFP), both of which were previously shown to

accumulate in aggresomes following treatment with MG132 or other

proteasome inhibitors (Engelender et al, 1999; O’Farrell et al, 2001;

Tanaka et al, 2004; Zaarur et al, 2008).

In the screen, we used HeLa cells co-expressing RFP-Ub and Syn-

GFP (Fig 1B). The entire siRNA library of the human genome was

screened. For each gene, we used a SMARTpool, that is a mix of

four different targeting siRNAs (Fig 1A). After siRNA transfections,

cells were treated with MG132 for 5 h, and the presence of aggre-

somes in different wells was evaluated by high-density microscopy

followed by image analysis (see Materials and Methods). We scored

various phenotypes, including: (i) the absence of an aggresome, (ii)

large multiple aggregates, and (iii) a smaller aggresome. We identi-

fied 425 hits that inhibited aggresome formation in more than 50%

of the cells in at least two out of three replicates. To validate the

hits, we rescreened the hit siRNA pools, by adding each siRNA oligo

from the hit SMARTpool individually in a separate well. Different

oligos from the same SMARTpool were dispensed into distant wells

to avoid local effects. Only hits that showed the inhibition of aggre-

some formation by at least two different oligos (out of 4) in all three

replicates was considered validated. By this approach, we elimi-

nated off-target effects. Images of all hit wells were rechecked

manually to confirm the results of the computer-based image analy-

sis. Our validation procedure reduced the list to 164 hits (Table 1),

among which 29 gene knockdowns demonstrated aggresome inhibi-

tion with all four siRNA sequences.

We used a gene functional classification tool from DAVID bio-

informatics software/database (http://david.abcc.ncifcrf.gov), to

classify the validated hit genes. Using the gene ontology category-

biological process, we identified seven main groups in our gene list

(Fig 1C). The largest functional group (24% of the validated genes)

was composed of genes involved in RNA processing. The second

largest group was involved in the cell cycle (13%). Of note, almost

half of this functional group was made up of genes involved in the

proteasome-ubiquitin system.

Surprisingly, a relatively low fraction of the identified genes was

involved in cytoskeleton dynamics or binding unfolded proteins.

Among genes involved in cytoskeleton-dependent transport, we

detected several major components of the dynein motor complex,

including DYNC1H1 (dynein, cytoplasmic 1, heavy chain 1),

DNC1I2 (dynein, cytoplasmic 1, intermediate chain 2), PAFAH1B1

(dynein motor regulator, Lis1), TUBB (tubulin, beta), and ACTR1A

(ARP1, actin-related protein 1 homolog A, centractin alpha)

(Fig 1D). The presence of these “expected” genes in our list

supported the overall validity of the screen and, in addition, identi-

fied specific isoforms of components of the dynein motor complex

involved in aggresome formation.

Depletion of RuvbL1 or RuvbL2 suppresses aggresome formationin mammalian cells

RuvbL1 and RuvbL2, also known as pontin and reptin, respectively,

were two of the 29 genes in which all four siRNA sequences we

tested suppressed aggresome formation (Appendix Fig S1). Their

activity was therefore not due to off-target effects. These proteins

belong to a diverse protein family of AAA+ ATPases, which among

other proteins include molecular chaperones, like Hsp104 or ClpB

(Doyle & Wickner, 2009; Winkler et al, 2012; Clare & Saibil, 2013).

Several reports suggested that RuvbL associates with a number of

multi-protein complexes, such as telomerase, certain RNPs, mTOR,

and related kinases, and serves in their assembly, thus suggesting a

putative chaperone function (Nano & Houry, 2013). We therefore

assessed the role of RuvbL in protein aggregation and aggresome

formation.

The EMBO Journal ª 2015 The Authors

The EMBO Journal RuvbL1/2 controls aggregation and aggresome Nava Zaarur et al

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We manually repeated tests to validate the effects of RuvbL

knockdowns using higher microscope magnification. HeLa cells

expressing Syn-GFP were depleted of either RuvbL1 or 2 using

individual siRNA, and the formation of aggresomes following

proteasome inhibition was assessed as in Zaarur et al (2008).

siRNAs against RuvbL1 or RuvbL2 suppress aggresome formation

by 60–70% (Fig 2A). A similar effect was observed with MCF10A

cells (Appendix Fig S2). Effect of RuvbL1 depletion was reversed by

expressing the siRNA-resistant version of recombinant RuvbL1

(Fig 2B and C). Interestingly, expression of the siRNA-resistant

mutant that carries two ATPase-inactivating mutations (A908G and

C915T) was ineffective (Fig 2B and C). Furthermore, unlike overex-

pression of normal RuvbL1, overexpression of the ATPase mutant

RuvbL1 partially inhibited aggresome formation (Fig 2D) and

therefore had a dominant-negative effect, further indicating that the

ATPase activity of RuvbL1 plays an important role in aggresome

formation.

Suppression of aggresome formation by depletion of RuvbL1

or RuvbL2 was also seen with RFP-Ub-decorated endogenous

polypeptides (Fig 2E). Of note, RuvbL1 depletion also reduced

recruitment of endogenous ubiquitin to the aggresome (Fig 2F). To

test whether these effects could be generalized, we conducted a

similar experiment with a different aggresome substrate, VHL-RFP;

recruitment of this polypeptide to the aggresome was strongly

suppressed by depletion of RuvbL1 (Fig 2G). Overall, these experi-

ments indicate that both RuvbL proteins are critical for aggresome

formation by a wide variety of substrates, including misfolded

polypeptides.

In its function in aggresome formation, RuvbL may act indirectly

by regulating expression of major molecular chaperones. We moni-

tored the activity of the heat-shock transcription factor Hsf1 using a

luciferase reporter (Kim et al, 2012) upon depletion of RuvbL1 or

RuvbL2. HeLa cells were infected with lentivirus encoding luciferase

under the control of HSE element in the promoter, and levels of luci-

ferase were measured in control and RuvbL-depleted cells. No

significant difference was observed (Fig 2H), indicating that RuvbL

does not play a role in heat-shock response. Similarly, depletion of

RuvbL proteins did not alter expression levels of the major chaper-

one Hsp70 (Fig 2I). Also, the effects of RuvbL depletion could not

be explained by hypothetical influence of RuvbL on protein degrada-

tion, since they could be seen upon inhibition of both proteasome

and autophagy (see, for example, Fig 6D).

Depletion of RuvbL2 leads to dramatic downregulation of

RuvbL1 (Fig 2J). Mutual regulation of RuvbL1 and RuvbL2 expres-

sion has been reported previously (Venteicher et al, 2008; Izumi

et al, 2010). Notably, overexpression of FLAG-tagged RuvbL1 in the

A

C D

B

Figure 1. High-throughput siRNA screening for aggresome formation.

A A scheme of the siRNA screening procedure as described in Materials and Methods.B Positive and negative controls from the screen. HeLa cells expressing synphilin-GFP and RFP-Ub were transfected with si-control (using anti-luciferase sequence) and

treated with 10 lM MG132 or left untreated. Scale bar, 10 lm.C Validated hits are categorized by gene ontology category-biological process, using “DAVID” bioinformatics resource (http://david.abcc.ncifcrf.gov/).D Defects in aggresome formation caused by siRNA against genes involved in microtubular transport. All cells were treated with 10 lM MG132. Scale bar, 10 lm.

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Table 1. Screen hit genes. siRNA that show inhibition of aggresome inmore than 30% of the cells and with more than two differentsequences of the siRNA.

Symbol Category Entrez gene name

ABLIM1 8 Actin-binding LIM protein 1

ACTN4 8 Actinin, alpha 4

ACTR1A 8 ARP1 actin-related protein 1 homolog A,centractin alpha (yeast)

ADD2 8 Adducin 2 (beta)

AHSP 7 Alpha hemoglobin-stabilizing protein

AKAP11 8 A kinase (PRKA) anchor protein 11

APOM 2 Apolipoprotein M

ARHGAP9 8 Rho GTPase-activating protein 9

ARSG 8 Arylsulfatase G

ATG9A 8 ATG9 autophagy related 9 homolog A(S. cerevisiae)

BCL2L10 8 BCL2-like 10 (apoptosis facilitator)

BMP7 8 Bone morphogenetic protein 7

BRF1 4,6 BRF1 homolog, subunit of RNA polymeraseIII transcription initiation factor IIIB(S. cerevisiae)

BTF3 6 Basic transcription factor 3

C2orf66 8 Chromosome 2 open reading frame 66

CAD 8 Carbamoyl-phosphate synthetase 2,aspartate transcarbamylase, anddihydroorotase

CCT4 7 Chaperonin containing TCP1, subunit 4(delta)

CCT8 7 Chaperonin containing TCP1, subunit 8(theta)

CDC40 1 Cell division cycle 40 homolog (S. cerevisiae)

CDC5L 1,3 CDC5 cell division cycle 5-like (S. pombe)

CENPH 3 Centromere protein H

CLTB 8 Clathrin, light chain B

CORT 8 Cortistatin

CRNKL1 1 Crooked neck pre-mRNA splicing factor-like1 (Drosophila)

CSNK2B 8 Casein kinase 2, beta polypeptide

CWC15 1 CWC15 spliceosome-associated proteinhomolog (S. cerevisiae)

CWC22 1 CWC22 spliceosome-associated proteinhomolog (S. cerevisiae)

DDX24 8 DEAD (Asp-Glu-Ala-Asp) box polypeptide 24

DHX8 1 DEAH (Asp-Glu-Ala-His) box polypeptide 8

DYNC1H1 3,5 Dynein, cytoplasmic 1, heavy chain 1

DYNC1I2 5 Dynein, cytoplasmic 1, intermediate chain 2

EFTUD2 1 Elongation factor Tu GTP binding domaincontaining 2

EIF3D 4 Eukaryotic translation initiation factor 3,subunit D

EXOSC8 1 Exosome component 8

Table 1 (continued)


FAM135B 8 Family with sequence similarity 135,member B

FGF12 8 Fibroblast growth factor 12

GAS2 3 Growth arrest-specific 2

GIMAP5 8 GTPase, IMAP family member 5

GPR12 8 G protein-coupled receptor 12

GPR153 8 G protein-coupled receptor 153

GRB14 8 Growth factor receptor-bound protein 14

GRIK3 8 Glutamate receptor, ionotropic, kainate 3

HGS 8 Hepatocyte growth factor-regulatedtyrosine kinase substrate

HIST1H3A 8 Histone cluster 1, H3a

HNRNPC 1 Heterogeneous nuclear ribonucleoprotein C(C1/C2)

HNRNPK 1 Heterogeneous nuclear ribonucleoprotein K

HOXB8 8 Homeobox B8

HSPA8 7 Heat-shock 70 kDa protein 8

IGLL1 8 Immunoglobulin lambda-like polypeptide 1

IGSF10 8 Immunoglobulin superfamily, member 10

IL17A 8 Interleukin 17A

INTS4 1 Integrator complex subunit 4

IRF3 6 Interferon regulatory factor 3

ISY1 1 ISY1 splicing factor homolog (S. cerevisiae)

KIF20A 5 Kinesin family member 20A

KRT81 8 Keratin 81

LEPROT 8 Leptin receptor overlapping transcript

LOC286149 8 Hypothetical protein LOC286149

LRP8 8 Low-density lipoprotein receptor-relatedprotein 8, apolipoprotein e receptor

LTBR 8 Lymphotoxin beta receptor (TNFRsuperfamily, member 3)

MAF1 8 MAF1 homolog (S. cerevisiae)

MAK16 8 MAK16 homolog (S. cerevisiae)

MC3R 8 Melanocortin 3 receptor

MFAP1 8 Microfibrillar-associated protein 1

MOBKL2A 8 MOB1, Mps One Binder kinase activator-like2A (yeast)

MUSTN1 8 Musculoskeletal, embryonic nuclearprotein 1

MYST1 4 MYST histone acetyltransferase 1

NACA 4 Nascent polypeptide-associated complexalpha subunit

NCKAP5L 8 NCK-associated protein 5-like

NEK11 3 NIMA (never in mitosis gene a)- relatedkinase 11

NLE1 8 Notchless homolog 1 (Drosophila)

ODC1 4 Ornithine decarboxylase 1



4


Table 1 (continued)


PABPN1 1 Poly(A) binding protein, nuclear 1

PAFAH1B1 5,3 Platelet-activating factor acetylhydrolase 1b,regulatory subunit 1 (45 kDa)

PANK4 8 Pantothenate kinase 4

PAXIP1 8 PAX interacting (with transcription-activation domain) protein 1

PCNT 3,5 Pericentrin

PDE8B 8 Phosphodiesterase 8B

PGBD3 8 PiggyBac transposable element derived 3

PHF5A 1 PHD finger protein 5A

POLR2F 1,6 Polymerase (RNA) II (DNA directed)polypeptide F

POLR2G 1,6 Polymerase (RNA) II (DNA directed)polypeptide G

PRODH2 8 Proline dehydrogenase (oxidase) 2

PRPF18 1 PRP18 pre-mRNA processing factor 18homolog (S. cerevisiae)

PRPF31 1 PRP31 pre-mRNA processing factor 31homolog (S. cerevisiae)

PRPF40A 1 PRP40 pre-mRNA processing factor 40homolog A (S. cerevisiae)

PRPF4B 1 PRP4 pre-mRNA processing factor 4homolog B (yeast)

PSMA1 2,3 Proteasome (prosome, macropain) subunit,alpha type, 1



PSMB1 2,3 Proteasome (prosome, macropain) subunit,beta type, 1

PSMB3 2,3 Proteasome (prosome, macropain) subunit,beta type, 3

PSMC1 2,3 Proteasome (prosome, macropain) 26Ssubunit, ATPase, 1



PSMD1 2,3 Proteasome (prosome, macropain) 26Ssubunit, non-ATPase, 1

PSMD3 2,3 Proteasome (prosome, macropain) 26Ssubunit, non-ATPase, 3

PTGR1 8 Prostaglandin reductase 1

PTK2 5 PTK2 protein tyrosine kinase 2

PUS7 1 Pseudouridylate synthase 7 homolog(S. cerevisiae)

PVRL3 8 Poliovirus receptor-related 3

RASSF4 3 Ras association (RalGDS/AF-6) domainfamily member 4

RPL13 4 Ribosomal protein L13

Table 1 (continued)






RPLP2 4 Ribosomal protein, large, P2

RPP25 1 Ribonuclease P/MRP 25 kDa subunit

RRM1 1,6 Ribonucleotide reductase M1

RSL24D1 4 Ribosomal L24 domain containing 1

RUVBL1 3,4 RuvB-like 1 (E. coli)

RUVBL2 3,4 RuvB-like 2 (E. coli)

SALL1 8 Sal-like 1 (Drosophila)

SAP30BP 8 SAP30 binding protein

SART1 1,3 Squamous cell carcinoma antigenrecognized by T cells

SETD8 3 SET domain containing (lysinemethyltransferase) 8

SF3A1 1 Splicing factor 3a, subunit 1, 120 kDa



SF3B1 3 Splicing factor 3b, subunit 1, 155 kDa

SF3B14 3 Splicing factor 3B, 14 kDa subunit





SHB 8 Src homology 2 domain containing adaptorprotein B

SLC25A10 8 Solute carrier family 25 (mitochondrialcarrier; dicarboxylate transporter),member 10

SLC48A1 8 Solute carrier family 48 (heme transporter),member 1

SLC9A3R2 8 Solute carrier family 9 (sodium/hydrogenexchanger), member 3 regulator 2

SMARCB1 3 SWI/SNF-related, matrix-associated, actin-dependent regulator of chromatin,subfamily b

SNCAIP 1 Synuclein, alpha interacting protein

SNRNP200 1 Small nuclear ribonucleoprotein 200 kDa(U5)

SNRNP70 1 Small nuclear ribonucleoprotein 70 kDa (U1)

SNRPA1 1 Small nuclear ribonucleoproteinpolypeptide A’

SNRPB 1 Small nuclear ribonucleoproteinpolypeptides B and B1

SNRPC 1 Small nuclear ribonucleoproteinpolypeptide C

SNRPD1 1 Small nuclear ribonucleoprotein D1polypeptide 16 kDa



5


RuvbL2-depleted cells (restoring the endogenous levels of RuvbL1,

as seen on Fig 2J, upper panel) did not restore aggresome formation

(Fig 2J, lower panel). Likewise, overexpression of RuvbL2 did not

restore aggresome formation in RuvbL1-depleted cells (Appendix

Fig S3). The fact that depleting either RuvbL1 or RuvbL2 decreased

aggresome formation suggests that the two proteins work in

complex in this function, which is consistent with the literature data

showing that RuvbL1 and RuvbL2 function as a mixed dodecamer

barrel-like structure (Gorynia et al, 2011; Tosi et al, 2013; Lakomek

et al, 2015).

In assembly/remodeling of multiprotein structures, RuvbL often

cooperates with other factors, forming so-called R2TP complex,

which involves Hsp90. However, inhibition of Hsp90 by a small

molecule 17-AAG did not mimic effect of RuvbL depletion on aggre-

some formation. In contrast, 17-AAG triggered aggresome and

enhanced aggresome formation when combined with MG132

(Fig EV1A). Furthermore, using the siRNA approach, we depleted

another major component of the R2TP complex Pih1 and did not

observe any effect on aggresome formation (Fig EV1B), indicating

that in this process, RuvbL proteins work either alone or by forming

other functional complexes.

Interaction of RuvbL with aggresome substrates

In a separate line of investigation, we used tandem affinity purifica-

tion in combination with label-free quantitative mass spectrometry

to identify proteins that associate with an aggresome substrate

synphilin-1 (see Materials and Methods). We utilized a modified

version of the minimal region of synphilin-1 that preserves aggrega-

tion and aggresome targeting properties of synphilin-1 (Fig 3E),

consisting of the ankyrin repeat domain ANK1, followed by the coil-

coiled (CC) and ANK2 domain (ANK1-CC-ANK2) (Zaarur et al,

2008). The construct was expressed in HEK293 cells, and these cells

and appropriate control cells were treated with and without protea-

some inhibitor MG132 for 4 h. After tandem affinity purification, the

samples were digested with trypsin and LC-MS/MS was performed

to identify proteins associated with synphilin-1, either directly or

through other proteins. Importantly, both RuvbL1 and RuvbL2 were

identified among synphilin-1-associated proteins (Fig 3A). We con-

firmed this interaction by co-immunoprecipitation of RuvbL1 with

the synphilin-1-derived construct ANK1-CC-ANK2 (Fig 4A).

Table 1 (continued)


SNRPD2 1 Small nuclear ribonucleoprotein D2polypeptide 16.5 kDa

SNRPD3 1 Small nuclear ribonucleoprotein D3polypeptide 18 kDa

SNRPE 1 Small nuclear ribonucleoprotein polypeptide E

SSPO 8 SCO-spondin homolog (Bos taurus)

STK11 3 Serine/threonine kinase 11

SUGP1 8 SURP and G patch domain containing 1

SUPT6H 8 Suppressor of Ty 6 homolog (S. cerevisiae)

TARDBP 1,3,6 TAR DNA binding protein

TCEB2 6 Transcription elongation factor B (SIII),polypeptide 2 (18 kDa, elongin B)

TCP10 7 T-complex 10 homolog (mouse)

TRHR 8 Thyrotropin-releasing hormone receptor

TUBB 3,5 Tubulin, beta

U2AF1 1 U2 small nuclear RNA auxiliary factor 1

U2AF2 1 U2 small nuclear RNA auxiliary factor 2

USP9X 3 Ubiquitin specific peptidase 9, X-linked

VPRBP 8 Vpr (HIV-1) binding protein

XAB2 1 XPA binding protein 2

ZNF124 8 Zinc finger protein 124




Genes categorized to biological process groups #1—RNA processing, #2—Proteasome-ubiquitin system, #3—Cell cycle, #4—Translation, #5—Microtubules-based process, #6—Transcription, #7—Unfolded proteinbinding, #8—Uncategorized.

▸Figure 2. RuvBL1 and RuvBL2 depletion block aggresome formation following proteasome inhibition.

A Effects of RuvbL1 or RuvbL2 depletion on recruitment of synphilin-GFP to aggresome. HeLa cells expressing synphilin-GFP were transfected with si-control, si-RuvbL1,or si-RuvbL2. After 72 h, cells were incubated with 5 lM of MG132 for 4 h. Images were taken with 100×magnification. Right panel shows quantification of these data.

B, C Expression of siRNA-resistant RuvbL1 but not the ATPase mutant reverses the effect of siRNA against RuvbL1. HeLa cells expressing synphilin-GFP were transfectedwith si-control or si-RuvbL1 and with the siRNA-resistant constructs (see Material and Methods) as mentioned or with control (empty vector). Cells were incubatedwith MG132 and the fraction of cells with aggresome was counted (B). Levels of expression of endogenous RuvbL1, WT, and mutant recombinant RuvbL1 in thisexperiment are shown in (C).

D Overexpression of the ATPase mutant of RuvbL1 leads to suppression of aggresome formation. Normal RuvbL1 does not show the dominant-negative effect.E Effects of RuvbL1 or RuvbL2 depletion on recruitment of RFP-Ub to aggresome in HeLa cells. HeLa cells expressing RFP-Ub were treated as in (A).F Effects of RuvbL1 depletion on recruitment of endogenous ubiquitin to aggresome in HeLa cells. RuvbL1-depleted and control HeLa cells were treated with 5 lM

MG132 for 5 h, and cells were fixed and subjected to immunostaining with anti-ubiquitin antibody.G Effects of RuvbL1 depletion on recruitment of VHL-Cherry to aggresome. Control and RuvbL-depleted HeLa cells were transfected with VHL-Cherry, and 24 h later,

cells were treated with 5 lM MG132 for 5 h.H RuvbL1 and RuvbL2 do not affect the heat-shock response. Cells expressing reporter luciferase under the control of HSE (heat-shock element) were transfected with

si-control and si-RuvBL1 or si-RuvbL2 and incubated with and without 10 lM MG132 for 2 h. Cells were lysed and luciferase activity was measured.I Control, RuvBL1- and RuvbL2-depleted cells were incubated with and without MG132 for 6 h, and the levels of Hsp70 were measured by immunoblotting.J RuvbL1 does not compensate for the aggresome defect following RuvbL2 depletion. RuvbL2 was depleted in HeLa cells expressing synphilin-GFP, and recombinant

RuvbL1 was overexpressed using transient transfection. The fraction of cells with aggresome was counted, and samples were subjected to immunoblotting withanti-RuvbL1 antibody (upper panel). Effects of RuvbL1 overexpression on aggresome is shown on lower panel.

Data information: All scale bars, 10 lm. In the aggresome quantifications, 200 cells were counted in each experiment. Experiments were repeated three times. The errorbars represent standard deviations. *P < 0.05, **P < 0.01.



6


A

B

E

F

H

I

J

G

C D

Figure 2.



7


To identify direct interactors with synphilin-1, we combined

mass spectrometry with chemical crosslinking (Fig 3B). ANK1-CC-

ANK2 with associated proteins was isolated from HEK293 cells, as

described above, and crosslinked on the affinity beads using BS3-

H12/D12 (bissulfosuccinimidylsuberate). The cross-linked proteins

were eluted from the affinity beads, digested by trypsin, and

analyzed by LC-MS/MS. The crosslinking analysis indicated that

RuvbL1 directly interacts with synphilin-1, and located the interac-

tion site, since synphilin-1K663 was linked to RuvbL1K372 (Fig 3C).

This was the only site of crosslinking between these two proteins.

We mapped the site involved in this interaction on the 3D structure

of RuvbL1 and determined that RuvbL1 interacts with synphilin

through one of its side chains, located on the surface of the ATPase

core and in proximity to the central channel (Fig 3D). Interestingly,

K663 residue critical for interaction with RuvbL1 is located in the

ankyrin repeat domain (ANK2) of synphilin-1 (Fig 3E, see arrow),

which is responsible for its aggregation (Zaarur et al, 2008). We

further built upon the uncovered structural information by testing

whether interaction with the substrate is important for the effect of

RuvbL on aggresome formation.

Functional importance of interaction of RuvbL with substratesfor aggresome formation

Previously, we demonstrated that the CC and ANK2 domains of

synphilin-1 are responsible for aggregation of this protein, while the

adjacent ANK1 domain is necessary for its aggresome targeting

(Zaarur et al, 2008). Accordingly, deletion of either CC or ANK2

from the ANK1-CC-ANK2 construct led to significantly reduced

aggregation. Yet, due to the presence of the ANK1 domain, either

deletion construct could be targeted to the aggresome, though signif-

icantly less efficiently than constructs that have all three domains.

Since the RuvbL binding site is located in the ANK2 region (see

above), first, we confirmed that deletion of the ANK2 region indeed

eliminated the association of RuvbL1 with the construct (Fig 4A).

Next, we asked whether the deletion construct lacking this region

and thus unable to bind to RuvbL remains sensitive to its depletion.

To test the role of synphilin-RuvbL interaction in aggresome

formation, HEK293 cells expressing ANK1-CC-ANK2-GFP and

ANK1-CC-GFP constructs were depleted of RuvbL1 and treated with

MG132 for 7 h. Upon the depletion, the efficiency of aggresome

formation by the full-length and ANK1-CC-ANK2-GFP constructs

dropped from 85 to 25%. On the other hand, the ANK1-CC-GFP

construct formed aggresomes in about 25% of control cells, and

RuvbL1 depletion did not significantly affect this fraction (Fig 4B).

Therefore, deletion of the RuvbL-interacting region from synphilin-1

decreased aggresome formation and made it insensitive to RuvbL,

demonstrating that interaction between synphilin-1 and RuvbL is

critical for efficient aggresome targeting of synphilin-1. These data

further support direct role of RuvbL proteins in the assembly and/or

disassembly of the aggresome.

We further tested whether RuvbL localizes to the aggresome.

FLAG-tagged RuvbL1 or HA-tagged RuvbL2 was co-expressed with

synphilin-GFP in HeLa cells, and the cells were treated with

MG132 for 2 h. In contrast to the diffuse distribution of RuvbL

proteins throughout the cytoplasm and nucleus in naı̈ve cells

(Appendix Fig S4), both RuvbL1 and RuvbL2 were recruited to

aggresomes following proteasome inhibition (Fig 4C), which was

consistent with their involvement in aggresome assembly and/or

disassembly. Furthermore, when transport of aggregates to the

aggresome was blocked by nocodazole, RuvbL localized to multi-

ple aggregates formed throughout the cytosol (Fig 4D), suggesting

that it is recruited to protein aggregates in the process of aggre-

some formation.

Direct association with aggresome and direct effect on aggresome

assembly or disassembly suggests that RuvbL may play a role in

protein aggregation or disaggregation. Indeed, microscopic observa-

tion of mammalian cells depleted of RuvbL and expressing either

Syn-GFP or VHL-GFP upon proteasome inhibition revealed that

many cells formed multiple small cytoplasmic aggregates that were

not recruited to aggresome (see Fig 2). To test for the effects of

RuvbL on the extent of protein aggregation, control and RuvbL1- or

RuvbL2-depleted HeLa cells expressing Syn-GFP were treated

with MG132. Cell lysates were fractionated by centrifugation at

13,000 g, and amounts of Syn-GFP in pellets and supernatants were

measured by immunoblotting. In the RuvbL1-depleted cells (both in

the presence and in the absence of MG132), the presence of Syn-

GFP in the pellet relative to supernatant was increased by about

two-fold (Fig 4E). Therefore, suppression of aggresome formation in

the RuvbL-depleted cells probably results from excessive protein

aggregation. These aggregates are either so large or so numerous

that they overwhelm the aggresome machinery. Such excessive

aggregation could result from a reduced ability of cells to refold or

disaggregate abnormal proteins. Taking into consideration the fact

that RuvbL proteins belong to the AAA+ protein family and that they

are involved in assembly of many protein complexes, these data

Figure 3. RuvbL1 and RuvBL2 interact with synphilin-1 directly.

A His-ANK1-CC-ANK2-GFP was expressed in HEK293 cells and isolated using cobalt affinity column, followed by anti-GFP antibody affinity column. Samples weretrypsin-digested and subjected to LC-MS/MS analysis. Results were analyzed, identified, and quantified using Progenesis LCMS and Mascot. The following RuvbL1peptides were identified: Y405-K418 (shown), and A318-R333, E47-K57, V358-K372, M61-K76, T401-K418, K60-K76, R118-K125, V91-K108, and T109-R123 (not shown).RuvbL2 peptides: V428-R437 (shown), A314-R329, G29-R39, T164-K183, Q40-R52, T115-R124, and S438-K455 (not shown).

B Work flow of determination of synphilin-1 binary interactions using isotopically labeled cross-linking and mass spectrometry.C MS2 spectra of the cross-linked peptide between synphilin-1 and RuvbL1. The orange peaks show the MS2 spectrum of the BS3-H12 cross-linked peptide, while

purple peaks show the MS2 spectrum of the BS3-D12 cross-linked peptide. The isotopically tagged cross-linkers were used to facilitate the cross-linked peptideidentification. The a peptide chain is the peptide R660-R666 from SNCAP (synphilin-1), and the b peptide chain is the peptide T363-K376 from RUVB1 (RuvbL1). Theions are classed as b ions when the charges are retained on the N-terminal fragment, and the ions are classed as y ions when the charged are retained on C-terminalfragment. Fragment ions without the cross-linker are labeled in green, and fragment ions with the cross-linker are labeled in red. The cross-linking sites weredetermined as the K663 residue on synphilin-1 and the K372 residue on RuvbL1.

D The cross-linking site on RuvbL1K372 is located at the surface of the barrel structure. The image was re-constructed from human RuvbL1 crystal structure (Matiaset al, 2006) using Protein Workshop (www.rcsb.org).

E The cross-linked site on synphilin-1 K663 is located at its ANK2 domain.

▸



8


http://www.rcsb.org

suggest that RuvbL may serve a chaperone function in the assembly

or disassembly of protein aggregates.

A

C

E

B

D

Figure 3.



9


Protein-stimulated ATPase activity of RuvbL

ATPase activity of the chaperones can be stimulated by their

polypeptide substrates, usually measured with model polypeptides,

such as casein (Woo et al, 1992; Schirmer & Lindquist, 1997).

Accordingly, we tested whether ATPase of RuvbL could be stimu-

lated by these substrates. Dodecamer RuvbL complex consisting of

two heterohexameric rings with alternating RuvbL1 and RuvbL2

monomers was purified as previously described (Gorynia et al,

2011) and used in the in vitro ATPase assay. Indeed, casein signifi-

cantly stimulated the ATPase activity of the RuvbL complex in a

dose-dependent manner (Fig 5A).

It was recently shown that domain II of both RuvbL1 and

RuvbL2 is auto-inhibitory, and its deletion enhances the ATPase

activity (Gorynia et al, 2011). Here, we tested whether deletion of

domain II affects the protein stimulation of the ATPase activity.

Accordingly, we compared casein stimulation of the ATPase of

normal and mutant RuvbL complex with deletion of the domain II

in both RuvbL1 and RuvbL2. As seen in Fig 5A, removal of the

auto-inhibitory domain II indeed enhances the stimulation of the

ATPase activity by casein. Interestingly, the protein stimulation of

the ATPase activity could also be observed with monomeric

RuvbL1 or RuvbL2 that was purified as previously described

(Gorynia et al, 2011) (Fig 5B), suggesting that interaction with

A B

C

D

E

Figure 4. Association of RuvbL with substrates is important for aggresome formation.

A Deletion of the ANK2 domain blocks association of synphilin-1 with RuvbL1. HEK293 cells were transfected with Flag-RuvbL1 and either with GFP (Cont.), full-lengthsynphilin-GFP (FL), ANK1-CC-ANK2-GFP, or ANK1-CC-GFP constructs. Cells were lysed and subjected to immunoprecipitation with anti-GFP antibody. AssociatedRuvbL1 was detected by immunoblotting with anti-RuvBL1 antibody.

B Deletion of the ANK2 domain relieves the RuvbL1 dependence of the recruitment to aggresome. HEK293 cells si-control and si-RuvbL1 were transfected with theindicated synphilin-1 deletion constructs and were treated with MG132 for 3 h. Fraction of cells with aggresome was evaluated; error bars represent standarddeviation of three repeats. **P < 0.01.

C RuvbL1 and RuvbL2 colocalize with aggresomes. HeLa cells expressing synphilin-GFP were transfected either with Flag-RuvBL1 or HA-RuvBL2. Cells were incubatedwith MG132 for 6 h, treated with 0.5% Triton X-100 for 20 s and immediately fixed. The cells were immunostained with anti-HA and anti-Flag antibodies. Scale bar:10 lm.

D RuvbL1 co-localizes with protein aggregates when transport to aggresome is blocked by nocodazole. HeLa cells transfected with Flag-RuvbL1 were incubated with5 lM MG132 and 10 lM nocodazole for 2 h and then fixed and immunostained with anti-Flag antibody.

E Effect of RuvBL1 depletion on synphilin-1 aggregation. RuvBL1-depleted and control cells were incubated with and without MG132 for 4 h. Soluble proteins andaggregates were separated by centrifugation for 15 min at 13,000 g. Pellets were 3.5 times more concentrated compared to the supernatant. The samples wereblotted with anti-GFP antibody (upper panel). Results of a typical experiment are shown. In three independent experiments, bands were quantified with ImageJ, andthe ratio of synphilin in pellets and total fractions was calculated (lower panel). Error bars represent standard deviations of the repeats.



10


substrate proteins may not require an assembly of the heterodode-

cameric structure.

Insulin is another polypeptide that stimulated the ATPase activity

of Hsp104 (Woo et al, 1992; Schirmer & Lindquist, 1997), and simi-

larly, it stimulated the ATPase of the RuvbL complex (Fig 5C).

Insulin is an amyloidogenic protein that can effectively form

amyloid fibrils under low pH conditions (Groenning et al, 2009).

Importantly, insulin fibrils have shown significantly stronger stimu-

latory effects on the RuvbL ATPase activity compared to soluble

insulin (Fig 5C).

Further, we compared effects of an amyloidogenic peptide

Ab1–42 on the ATPase activity of the RuvbL complex. The peptide

monomer significantly stimulated the ATPase activity (Fig 5D).

Importantly, the preformed Ab amyloid fibrils stimulated the

ATPase activity much stronger (almost four-fold). It should be

noted, that during the time of measurement of the ATPase activity,

the Ab monomer has partially polymerized (Appendix Fig S5, see

also Fig 6A). Therefore, the ATPase stimulation by the monomer is

overestimated, and the difference between effects of the amyloid

and the monomer could be even stronger. Thus, both soluble and

aggregated proteins can interact with RuvbL complex and stimulate

its ATPase activity, and amyloid fibrils have stronger effects

compared with the unaggregated forms of the corresponding

polypeptides. These data are consistent with the chaperone function

of RuvbL and further indicate that direct interaction of substrates

with RuvbL1 is functionally important.

RuvbL-stimulated remodeling of protein aggregates

To address effects of RuvbL on dynamics of protein aggregation, we

assessed how purified dodecamer RuvbL complex affects formation

of Ab fibrils by measuring thioflavin fluorescence, which is

enhanced upon binding to b-sheets, formed within aggregates. The

first step of Ab amyloidogenesis, seeding of fibrils, is a slow process,

A

C

B

D

Figure 5. Protein-stimulated ATPase activity of RuvBL.

A Effect of casein on the ATPase activity of the normal and mutant (deletion of domain II) RuvbL complexes.B Casein can stimulate the ATPase activities of monomeric RuvbL1 and RuvbL2.C Effect of soluble insulin and insulin fibrils on the ATPase activity of the RuvbL complex (see Materials and Methods).D Effect of 1–42 Ab peptide and Ab fibrils on the ATPase activity of the RuvbL complex.

Data information: All experiments were repeated at least three times; error bars represent standard deviations of the repeats.



11


A

C

D

E

F G

B

Figure 6.



12


which is followed by a faster process of fibril growth. Addition of

RuvbL without ATP significantly delayed fibril seeding and reduced

the rate of fibril growth (Fig 6A). This effect could not be through

interaction of RuvbL with Ab monomers, since there was almost a

2,000× molar excess of monomers compared to the RuvbL complex.

It is likely that in the absence of ATP, RuvbL binds to oligomeric

seeds and fibrils and prevents their growth. Addition of ATP

reverses this effect of seeding suppression or fibril growth (Fig 6A

and B), suggesting that in the presence of ATP either interaction of

RuvbL with the seeds is reduced or seed multiplication is promoted

(both outcomes were seen with other chaperones). These scenarios

agree with the data pointing to direct interaction between RuvbL

and Ab. Of note, we could not investigate effects of RuvbL on

insulin fibril formation, since this process takes place at low pH

conditions, inactivating RuvbL.

Further, we tested whether RuvbL cooperates with Hsp70 and

Hsp40 (analogous to Hsp70/Hsp40/Hsp104 chaperone system) in

suppression of Ab fibril formation. In the absence of RuvbL, the

Hsp70/Hsp40 chaperone pair also delayed seeding and reduced the

rate of fibril growth. Addition of RuvbL did not further suppress fib-

ril formation (Fig EV2A). Therefore, in this reaction, RuvbL does

not cooperate with Hsp70/Hsp40, and furthermore because of the

lack of additivity, probably these chaperones interact with the same

sites on the seeds to suppress Ab polymerization. Also, stimulation

of ATPase activity of Hsp70/Hsp40 and RuvbL by casein is not addi-

tive (Fig EV2B).

To address activity of RuvbL in protein disaggregation, we used

preformed insulin fibrils (experiments with Ab fibrils were

complicated by instability of thioflavin fluorescence and fibril

heterogeneity). We evaluated fragmentation of insulin fibrils by

atomic force microscopy. The chaperone-dependent deconstruction

of insulin fibrils is known to start with fibril swelling, followed by

disintegration into shorter fragments or amorphous aggregates of

irregular size (Kurouski et al, 2012, 2013; Min et al, 2014). In

control samples without RuvbL complexes, fibrils remained intact

throughout the incubation period (Fig 6C). Incubation with RuvbL

in the absence of ATP led to significant swelling of fibrils indicating

association of RuvbL with the fibrils or partial loss of structure.

Importantly, incubation with RuvbL in the presence of ATP led to

conversion of the insulin fibrils into amorphous aggregates of irreg-

ular size, although some remaining swollen fibrils were still seen

(Fig 6C). Therefore, as with Ab, RuvbL interacted with insulin

fibrils even in the absence of ATP, and ATP addition stimulated

fibril remodeling.

To address the disaggregation effects of RuvbL in vivo, we

followed disappearance of the preformed cellular aggregates after

washout of the proteasome inhibitor. In order to exclude the auto-

phagic component in the disappearance of aggregates, the experiment

was done in the presence of the autophagy inhibitor bafilomycin

(see Materials and Methods). Under these conditions, the recovery

of aggregates was most likely due to remodeling followed by disag-

gregation, and then by either refolding or proteasome degradation

of solubilized protein molecules. An additional complexity of this

experiment is that unlike control cells that form single aggresome in

response to MG132, RuvbL-depleted cells form multiple aggregates

(see above). Therefore, to follow disappearance of comparable

aggregate structures, we blocked aggresome formation by the micro-

tubule inhibitor nocodazole. Control and RuvbL1-depleted cells

were incubated with nocodazole and MG132 for 2 h, which resulted

in formation of multiple aggregates in both cultures (Fig 6D). Then

MG132 and nocodazole were washed out, and disappearance of the

aggregates was followed microscopically. In control cells, upon

recovery from the inhibitors, multiple aggregates proceeded to

aggresome within 1–2 h in almost every cell. These aggresomes

then gradually disappeared within the next 2 h. However, the fate

of aggregates in the RuvbL-depleted cells was different: upon

removal of MG132 and nocodazole, aggresomes appeared much

slower than in control in only about 30% of cells, while multiple

aggregates remained in the rest of cells. Importantly, the disappear-

ance of aggregates from cells was not seen at least in the course of

5 h following the removal of MG132 (Fig 6D and E). Similar

suppression of disaggregation in RuvbL-depleted cells was seen with

ubiquitinated proteins (they co-localized with synphilin aggregates)

(Fig EV4).

To exclude aggregates recruitment into aggresome upon removal

of MG132 and nocodazole, we followed their disappearance in cells

depleted of dynein subunit DYNC1H1. In our screen, this depletion

resulted in strong aggresome suppression and accumulation of small

aggregates (Fig 1D). This alternative way to inhibit aggresome is

much less toxic than continued incubation with nocodazole after

removal of MG132. Accordingly, cells were transfected with

DYNC1H1 siRNA with or without RuvbL1 siRNA. Aggregates were

◀ Figure 6. RuvBL1/2 complex suppresses formation and promotes deconstruction of protein aggregates.

A RuvbL suppresses 1–42Ab fibrillation. RuvbL complex was incubated with Ab peptide and thioflavin with or without ATP for 8 h, and fluorescence was read every6 min.

B Seeding time, Tlag, and fibrillation rate, K1/2, were calculated by fitting sigmoidal equation.C Deconstruction of insulin fibril by the RuvbL complex was performed using atomic force microscopy (see Materials and Methods). AFM images showing swelling of

amyloid fibrils by RuvbL without ATP and further deconstruction in the presence of ATP. Scale bar, 500 nm.D, E Effects of RuvbL on aggregates during recovery from proteasome inhibition. Control and RuvBL1-depleted HeLa cells expressing synphilin-GFP were incubated

with 10 lM nocodazole and 5 lM MG132. After 2 h, cells were washed four times with PBS and incubated with 10 lM emetine and 1 lM bafilomycin for theindicated time periods. Time “0” is the time of the start of recovery. Scale bar, 10 lm. At indicated times, the cells with aggresome, multiple aggregates, anddiffused synphilin were counted and the quantification of the experiment is shown in (D). Cells with aggregates are cells with either aggresome or multipleaggregates.

F Effect of RuvbL on recovery of aggregates in the absence of aggresome. HeLa cells expressing synphilin-GFP were transfected with si-control and si-DYNC1H1 orwith si-RuvbL1 and si-DYNC1H1. After 72 h, cells were incubated with 10 lM MG132 for 2 h, followed by the washout of the inhibitor. Time “0” is the time of thestart of recovery in the presence of 10 lM emetine and 1 lM bafilomycin. For quantification, percentages of cells with aggregates were evaluated.

G Depletion of RuvbL1 sensitizes to proteotoxic stress. Cells were transfected with control siRNA or si-RuvbL1 and incubated with MG132 or AZC for 5 h followed by10 h of recovery. Viability of cells was measured by CellTiter 96 (Promega). The experiment was done in triplicates, error bars represent standard deviations. *P < 0.05.

Data information: In (E) and (F), 200 cells were counted in three different experiments. Error bars represent standard deviations of the repeats.



13


induced with MG132, and the inhibitor was removed to allow for

the disappearance of aggregates. This experiment demonstrated that

RuvbL1 depletion significantly reduced the rate of aggregate disap-

pearance in cells (Fig 6F), and thus, RuvbL is a chaperone involved

in disassembly of protein aggregates.

The role of RuvbL in the aggregate disassembly suggests that it

may provide protection from proteotoxic stresses. Accordingly,

depletion of RuvbL may make cells more sensitive to treatments that

cause proteotoxicity, like inhibition of the proteasome or incubation

with amino acid analogs, which incorporate into polypeptides and

prevent normal folding. To study effects of RuvbL on cell physiol-

ogy, we treated a neuron-related cell line SY5Y with 10 lM MG132

or 10 lM of a proline analog azetidine-2-carboxylate (AZC) for 5 h,

washed out these reagents, and assessed cell viability after 10-h

recovery. These treatments were very mild and did not cause toxic-

ity in control cells, while in RuvbL1-depleted cells, a significant toxi-

city was seen (Fig 6G). Therefore, in line with its chaperone

function, endogenous RuvbL plays a role in survival of proteotoxic

stress.

RuvbL orthologs influence aggregate toxicity and formation ofaggresome-like structures in yeast

We tested whether yeast orthologs of RuvbL1 and 2 (respectively,

Rvb1 and Rvb2) are involved in the formation of aggresome-like

structures in yeast. To study protein aggregation, we chose a widely

used model of polyglutamine-containing polypeptide 103QP, which

we developed in the past (Meriin et al, 2002; Wang et al, 2009).

The 103QP construct includes exon 1 of the human huntingtin

protein with the expanded polyQ region, corresponding to the

severe form of Huntington’s disease. Due to the presence of

the proline-rich (P) region immediately following the polyQ region,

the 103QP protein is sequestered into an aggresome-like body

(Wang et al, 2009), a process similar to the recruitment of amyloid

aggregates into the IPOD compartment (Kaganovich et al, 2008).

First, we tested whether Rvb1 and Rvb2 proteins colocalize with

the aggresome-like body in yeast. Accordingly, we employed strains

in which endogenous RVB1 and RVB2 genes were fused with the

GFP ORF. These strains have been transformed with the plasmid

bearing 103QP-RFP. Indeed, both Rvb1-GFP and Rvb2-GFP localized

at single bodies in a significant fraction of yeast cells, and these

bodies colocalized with 103QP-RFP aggresome-like structures in

more than half of the cells (Fig 7A). These data indicated that, like

their mammalian counterparts, Rvb1 and Rvb2 proteins are local-

ized to aggresome-like bodies in yeast, which is consistent with their

role in aggresome formation.

To investigate the effects of Rvb1 and Rvb2 on formation of the

aggresome-like bodies, we depleted either of these proteins in yeast.

Since both RVB1 and RVB2 are essential for cell viability, we have

employed yeast strains in which a single copy of either gene is

placed under the control of the tetracycline-regulated promoter

(PTET). In these strains, expression of a particular gene can be down-

regulated by adding the tetracycline-related antibiotic doxycycline.

We used it at concentrations below 100 ng/ml to allow partial

depletion of the corresponding proteins without growth inhibition

(Appendix Fig S6). Microscopic observation showed that downregu-

lation of either RVB1 or RVB2 gene led to a significant reduction in

the proportion of cells containing large aggresome-like structures

with multiple aggregates being formed instead (Fig 7B and C). This

result indicates that similar to their mammalian counterparts, Rvb1

and Rvb2 proteins are involved in targeting misfolded proteins to

aggresome-like structures in yeast.

Previously, we demonstrated that the 103QP polypeptide usually

does not cause toxicity in yeast strains. However, either alteration of

protein sequestration patterns of 103QP (Wang et al, 2009; Gong

et al, 2012) or inhibition of the recruitment of 103QP to aggresome-

like bodies (Wang et al, 2009; Gong et al, 2012) triggered its toxicity.

In line with these findings, partial downregulation of either RVB1 or

RVB2, which suppressed targeting of 103QP to the aggresome-like

body, resulted in high toxicity of 103QP (Fig 7D). Notably, this

toxicity was caused by the expanded polyglutamine domain, since a

short polyglutamine polypeptide 25QP remained nontoxic. There-

fore, Rvb proteins appear to specifically control toxicity of poly-

peptides with expanded polyglutamine via regulation of their

aggregation status. Furthermore, consistent with their role

in proteostasis, mild downregulation of Rvb1 or Rvb2 increased

the sensitivity of yeast cells to the antibiotic hygromycin, which

causes accumulation of misfolded endogenous proteins in the cell

(Fig 7E).

According to the current paradigm, heat shock of cells causes

massive misfolding and aggregation of proteins, leading to cell

death, and the Hsp104 chaperone is the major factor counteracting

Figure 7. Rvb1 and Rvb2 influence protein aggregation and proteotoxicity in yeast.

A Rvb1-GFP and Rvb2-GFP colocalize with the aggresome-like structures in yeast. The Rvb1-GFP or Rvb2-GFP was co-expressed with 103QP-RFP.B Underexpression of Rvb1 or Rvb2 in the presence of moderate (in this case, 25 ng/ml) concentrations of doxycycline affects formation of the aggresome-like body in

cells expressing 103QP. 103QP was induced in the galactose medium for 6 h. Data obtained in a representative experiment are shown. Experiments were repeated 5times with variable concentrations of doxycycline. Numbers varied among the experiments, but effects of RVB downregulation relative to wild-type were similar.

C Appearance of the aggresome-like body and multiple aggregates.D Effects of RVB1 or RVB2 downregulation on sensitivity to protein aggregation. The 103QP construct becomes toxic to the PTET-RVB1 and PTET-RVB2 strains in the

presence of moderate concentrations (75 ng/ml) of doxycycline, which are not sufficient to inhibit growth in the presence of 25QP control. Growth inhibition by103QP is more severe in the PTET-RVB1 strain, compared to PTET-RVB2. All polyQ constructs were expressed from the galactose-inducible (PGAL) promoter.

E Downregulation of PTET-RVB1 or PTET-RVB2 by 25 ng/ml of doxycycline (Doxy) causes sensitivity to low concentration (20 lg/ml) of hygromycin (Hyg); in the sameconditions, growth of the wild-type (WT) strain is not inhibited.

F Exponential yeast cultures grown at 25°C and bearing multicopy plasmids with either RVB1 or RVB2 gene under their own promoters are more resistant to heat shockat 50°C, compared to the culture of the same strain bearing the control plasmid. Yeast cultures were plated onto �Ura medium, in order to monitor only plasmid-containing cells. Experiments were performed in triplicates.

G Excess Rvb1 or Rvb2 partially compensates for thermotolerance in the hsp104 deletion (hsp104Ä) mutant. Data shown are for the strains of BY series; major resultswere also reproduced for the strains of GT81 series (for strain description, see Appendix Supplementary Methods). Experiments were performed in triplicates.

Data information: Error bars represent standard deviations of the repeats.

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14


heat-shock-induced aggregation and cell death in yeast (Glover &

Lindquist, 1998). Hsp104 protein levels are extremely low in

exponentially growing cells at a normal temperature (25 or 30°C),

resulting in high sensitivity to heat shock (50°C). However,

overexpression of recombinant Hsp104 leads to thermotolerance.

Considering that Rvb may serve a chaperone function similar to

Hsp104, we asked whether Rvb overexpression might provide ther-

motolerance to exponential cells containing low levels of Hsp104.

Indeed, introduction of multicopy plasmids bearing RVB1 or RVB2

increased resistance to 50°C heat shock of yeast exponential cells

growing at 25°C (Fig 7F). Effect of RVB1 was the strongest (about

150-fold after 10-min exposure) and could be detected even when

expressed from a single-copy plasmid (data not shown).

Moreover, excess Rvb was able to reverse reduced thermotoler-

ance of the hsp104D strain (Fig 7G). Effects of Rvb1 or Rvb2

overexpression were similar to effects of restoration of Hsp104 on a

single-copy plasmid. These data indicate that Rvb proteins can

modulate thermotolerance independently of Hsp104. Therefore, Rvb

proteins expressed at high levels can at least partially substitute for

the Hsp104 function in thermotolerance. Together with data in

mammalian cells described above, these results support the function

for RuvbL/Rvb in protein homeostasis.

A

B

D

E F G

C

Figure 7.



15


Discussion

Here, we have developed a siRNA screen to identify factors involved

in aggresome formation, and RuvbL1 and RuvbL2 came up as strong

hits in the screen. Depleting both proteins reduced the fraction of

cells containing aggresomes and increased the fraction of cells with

multiple aggregates. This effect was observed with two aggregation-

prone polypeptides: synphilin-1 and VHL, as well as with endoge-

nous ubiquitylated proteins. These data suggested that (i) RuvbL

proteins play a role in control of protein aggregation, potentially as

a novel molecular chaperone and (ii) aggregates that are formed in

the absence of RuvbL are either too large to be transported to the

aggresome, or their overabundance overwhelms the aggresome

machinery. Interestingly, a chaperone function in assembly of

certain protein complexes has been proposed for mammalian RuvbL

(Nano & Houry, 2013). For example, they were suggested to partici-

pate in assembly of mTOR-like kinases, certain RNPs, telo-

merase, RNA polymerase (Venteicher et al, 2008; Izumi et al, 2011;

Horejsi et al, 2013; Kim et al, 2013; Machado-Pinilla et al, 2013),

and other complexes. Here, we report that RuvbL appears to be a

major player in general protein homeostasis.

The Hsp104 family members exist in bacteria, yeast, and plants,

where they facilitate protein disaggregation. However, they are not

found in metazoan (except mitochondria). The protein disaggregat-

ing activity existing in metazoa was attributed to the Hsp110-Hsp70-

DnaJ system, suggesting that there may be no AAA+ chaperone in

this taxonomy group that has functional analogy to Hsp104.

Indeed, mammalian Hsp110, Hsp70 (Hsc70 or Hsp70), and Hsp40

(Hdj1) were able to dissolve large protein aggregates and recover

natively folded proteins (Rampelt et al, 2012; Winkler et al, 2012;

Mattoo & Goloubinoff, 2013; Torrente & Shorter, 2014). On the other

hand, unlike Hsp104/Hsp70/Hsp40, this chaperone system failed to

disaggregate amyloids (Shorter, 2011). Our findings suggest that

RuvbL may constitute the missing AAA+ disaggregase in mamma-

lian and other metazoan cells.

Surprisingly, RuvbL expression was not induced under stressful

conditions such as heat shock or proteasome inhibition, both of

which lead to the buildup of abnormal protein species. Nor did

RuvbL depletion activate Hsf1 or cause accumulation of Hsps, two

other stress-related processes. Possibly, RuvbL becomes critical for

homeostasis only under a specific set of stressful conditions.

Previously, we demonstrated that in yeast, the model polyglu-

tamine-containing polypeptide 103QP (which corresponds to the

disease-causing exon 1 of huntingtin) accumulates in an aggresome-

like body (Wang et al, 2009), which may be the same as IPOD

compartment (Kaganovich et al, 2008). A similar polypeptide 103Q

missing the proline-rich region that follows the polyQ track could

not be recruited to this body, but instead formed multiple aggregates

in the cytoplasm that were toxic to yeast cells (Meriin et al, 2002,

2003). The formation of these multiple aggregates was dependent

on the presence of endogenous prions controlled by the AAA+ chap-

erone Hsp104, while recruitment of 103QP to the aggresome-like

body was not (Meriin et al, 2002; Wang et al, 2009). This current

study reports that yeast orthologs of RuvbL play a role in recruit-

ment of 103QP to the aggresome-like body, and downregulation of

these orthologs, which prevents the recruitment, leads to strong

103QP toxicity. These data indicate that though RuvbL may have

similar function in amyloid disassembly as Hsp104, these different

chaperones play distinct roles in recruitment to different aggregate

compartments.

A complex of data presented here strongly suggests that RuvbL

may serve as a chaperone in protein homeostasis. It includes accu-

mulation of protein aggregates upon RuvbL depletion, delayed

protein disaggregation in RuvbL-depleted cells, localization of

RuvbL to aggresomes, direct interaction of RuvbL with the aggre-

some substrate synphilin-1, stimulation of the ATPase activity of

RuvbL by misfolded proteins, and fragmentation of amyloid fibrils

in vitro. Furthermore, Ruvbl has a barrel-like structure with an

internal channel, showing certain structural similarity to common

chaperones, for example, ClpB or Hsp104 family members.

In protein disaggregation, Hsp104 cooperates with Hsp70 and

Hsp40. Here, we tested whether Hsp70/Hsp40 can similarly cooper-

ate with the RuvbL complex. A classical Hsp70/Hsp40/Hsp104

substrate luciferase could not be tested with RuvbL because of the

specificity of the latter. Indeed, RuvbL depletion did not affect luci-

ferase refolding in cells (Fig EV3), and purified RuvbL did not

stimulate luciferase refolding in vitro, indicating that luciferase is

not a RuvbL substrate. Therefore, we switched to proteins that can

interact with RuvbL. In these experiments, Hsp70/Hsp40 did not

promote ATPase activity of RuvbL-stimulated by casein (not

shown). Furthermore, Hsp70/Hsp40 did not show synergy or addi-

tivity with RuvbL in suppression of formation of Ab amyloid fibrils

(Appendix Fig S5). Finally, inhibition of Hsp70-Hsp40 interaction

with a small molecule myricetin did not affect disaggregation of

aggresome or synphilin-1 aggregates (Fig EV5). Together, these data

suggest that RuvbL does not cooperate with Hsp70/Hsp40 in its

chaperone function.

Interestingly, a function for mammalian RuvbL has been previ-

ously proposed in the assembly of certain protein complexes (Nano

& Houry, 2013), including mTOR-like kinases, certain RNPs, telo-

merase, and RNA polymerase (Venteicher et al, 2008; Izumi et al,

2011; Horejsi et al, 2013; Kim et al, 2013; Machado-Pinilla et al,

2013). In these cases, RuvbL often functions as part of the so-called

R2TP complex, which involves proteins Pih1 and Tah1 (Nano &

Houry, 2013), which connect it to Hsp90 and prefoldin (Boulon et al,

2012a,b; Mita et al, 2013). However, Hsp90 is unlikely to cooperate

with RuvbL, at least in aggresome formation, since inhibition of

Hsp90 does not mimic effects of RuvbL depletion of aggresome

(Fig EV1A). Furthermore, we did not see any effect of Pih1 depletion

on aggregation or aggresome formation (Fig EV1B). Prefoldin, on

the other hand, may be involved in delivery of substrates to the

RuvbL complex, similarly to its function in delivery of substrates to a

distinct chaperone TRiC (Hartl & Hayer-Hartl, 2002).

While DNA helicase activity of Rvb in bacteria was demonstrated

long ago, similar activity of mammalian RuvbL has not been found

until recently. Indeed, recent studies of mixed RuvbL1/2 dodecamer

structure indicated that the channel has a relatively monotonous

distribution of negative and positive charges, which was suggested

to facilitate interaction with single-stranded DNA molecules in order

to promote DNA helicase activity (Gorynia et al, 2011). On the other

hand, the charge distribution in the channel is also consistent with

binding of unfolded polypeptide chains that may be necessary for the

chaperone activity of RuvbL. We would like to emphasize, however,

that alternatively RuvbL may promote protein disaggregation not by

threading polypeptide chains through the channel, but rather by

interacting with hydrophobic surfaces or other patches on



16


aggregated proteins. Such interactions may be sufficient to shift the

equilibrium so that these polypeptides leave aggregates.

Materials and Methods

Reagents and antibodies

MG132, nocodazole, and bafilomycin were purchased from Biomol

(Farmingdale, NY, USA); Protease inhibitors tablets were from

Roche; Ni-NTA (Ni-nitrolotriacetate) superflow cartridge (1 × 5 ml)

was from Qiagen; MBP (Maltose Binding Protein) TrapTM HP

(1 × 1 ml) and Superdex G200 HR column (1 × 30 cm) were from

GE healthcare; Imidazole buffer solution was from Sigma.

Anti-tubulin antibodies (Abs) were from GenScript (Piscataway,

NJ, USA); anti-multi-ubiquitin Abs (FK2)—from Enzo Life Sciences

(Farmingdale, NY, USA); anti-RuvbL1 and anti-Flag Abs—from

Sigma-Aldrich (St. Louis, MO, USA); anti-HA Abs—from Cell Signal-

ing (Danvers, MA, USA); anti-GFP Abs—from Clontech (Mountain

View, CA, USA); and anti-actin Abs—from Santa Cruz (Santa Cruz,

CA, USA).

Constructs

The retroviral expression constructs with C-terminally tagged

synphilin-1 (Syn-GFP) and with mRFP-Ub were described before

(Zaarur et al, 2008, 2014). Synphilin deletion constructs ANK1-CC-

ANK2 and ANK1-CC were described before (Zaarur et al, 2008). For

tandem affinity purification, we employed the pEGFPN1 plasmid

with cloned a construct ANK1-CC-ANK2 with a His-tag at the

N-terminus and a GFP-tag at the C-terminus, as described previously

(Zaarur et al, 2008). VHL plasmid was a gift from Daniel Kaganovich

(Hebrew University, Israel). FLAG-RuvbL1 and HA-RuvbL2 were a

gift from Anindya Dutta (University of Virginia, US). For the rescue

experiment, the constructs of WT RuvbL1 and the RuvbL1 ATPase

mutant (A908G and C915T) were cloned into pCXIP and silence

mutations of A498C and C501A were introduced in both of them.

The pET21-N-ter-His6-TIP48 and pET15-His6-TIP49 plasmids coding

for RuvbL2 and RuvbL1, respectively, were a gift from I. Tsaneva.

Cells cultures, growth, and transfection

HeLa, HEK93, and SY5Y cells were grown in Dulbecco’s modified

Eagle’s medium (Gibco) supplemented with 10% fetal bovine serum

or heat-inactivated serum (for HEK93), at 37°C in an atmosphere

of 5% CO2. For fluorescence microscopy, cells were grown on

Lab-Tek� Chambered Coverglasses (NUNC) pretreated with poly-

L-lysine (Sigma). For transient plasmid transfection, we used

Lipofectamine 2000 reagent (Invitrogen), and for siRNA transfec-

tion, we used RNAiMAX reagent (Invitrogen).

siRNA screening

High-throughput siRNA screening was done using the screening

facility of the Institute of Chemistry and Cell Biology at Harvard

Medical School, ICCB. We screened 21,121 siRNA pools of four

different sequences for each gene, from the Dharmacon human

siGENOME Smart Pool, in triplicate. HeLa cells that express

Syn-GFP and RFP-Ub were reverse-transfected with the siRNA

pool. A 10-ll mixture of the lipid, Lipofectamine RNAiMAX (Invit-

rogen), diluted 1:20 with Optimem (Gibco), was dispensed auto-

matically to a 384-well black clear bottom plate (Corning 3712,

Corning, NY) using Matrix WellMate (Thermo Scientific). Immedi-

ately after, 2 ll of siRNAs were transferred from Dharmacon

library plate to the final concentration of 50 nM. As a negative

control, we used anti-luciferase siRNA, and for transfection effi-

ciency control, we used siRNAs against GFP (to assess disappear-

ance of Syn-GFP fluorescence) and against PLK1 (polo-like kinase 1;

depletion of which caused cell death). Each plate contained nega-

tive control and transfection control wells. Plates were briefly

centrifuged. After 30 min, 500 cells per well, diluted with 40 llDMEM, were dispensed to each well. Plates were kept in the incu-

bator for 72 h, for complete depletion. MG132 solution was added

to the wells at final concentration of 10 lM. Following 5 h of incu-

bation, cells were fixed with 4% formaldehyde and stain with

Hoechst 33342 (Sigma). Each plate was run in triplicate. Auto-

mated images using an ImageExpress Micro microscope (Molecular

Device) at 20× magnification at three wavelengths (for FITC, Texas

Red and DAPI) were taken for four fields per well. The percent of

synphilin and ubiquitin aggresome was determined by image anal-

ysis software written by Tiao Xie (ICCB) in Matlab. The screening

assay was very robust and yielded a z factor of 0.67. Because of

the diverse phenotype of aggresomes, confirmation of the hit was

done manually, by viewing the images.

Cell lysis and analysis

Cells were lysed with lysis buffer (40 mM Hepes, pH 7.5, 50 mM

KCl, 1% Triton X-100, 2 mM DTT, 1 mM Na3VO4, 50 mM b-glyc-erophosphate, 50 mM NaF, 5 mM EDTA, 5 mM EGTA, 1 mM

PMSF, protease inhibitor cocktail (Roche, Switzerland)). Samples

were adjusted to have equal amount of total protein and subjected

to electrophoresis followed by immunoblotting.

Immunostaining and microscopy

For immunostaining, cells were fixed for 10 min with 4% formalde-

hyde and washed with PBS. Then the cells were blocked for 1 h

with 3% BSA, and incubated at room temperature for 2 h with

primary antibody. Following a wash with PBS, cells were incubated

for an hour with Alexa Fluor 594 donkey anti-mouse IgG or anti-

rabbit (1:500) (Molecular Probes, Eugene, OR). After a final wash,

cells were analyzed with a fluorescence microscope, Axiovert 200

(Carl Zeiss, Germany) using a 100× oil objective. To assess the frac-

tion of cells with aggresome, at least 200 cells were counted in each

well. Live fluorescence microscopy of yeast cells containing the GFP

and/or RFP tags was performed under the fluorescence microscope

Olympus BX41, using a 100× oil objective.

Immunoprecipitation

Cells were grown on 60-cm plates and transfected with the

mentioned construct. After 24 h, cells were lysed with 30 mM NaCl,

10 mM Hepes, 0.5% Triton X-100, 5% glycine, 1.5 mM MgCl2, and

protease inhibitor cocktail (Roche, Switzerland). Sample from cell

lysate was kept as the “total”. For pull down, anti-GFP-Tag-Agarose



17


(MBL, Woburn, MA) was used, following the manufacturer’s

protocol.

Luciferase assay

Cells were infected with HSE-Luc lentivirus and GFP retrovirus.

Three days after selection, cells were heat-shocked at 45°C for

15 min followed by 3 h of incubation at 37°C. After the incubation

period, the medium was aspirated, cells were washed twice with

PBS, lysed, and transferred into 96-well plate. 50 ll of luciferase

assay reagent (Promega) was injected into each well, and lumines-

cence was read with a luminometer. In parallel, lysates were plated

into black 96-well plates and GFP fluorescence was read by the

same luminometer. All measurements were done in triplicate, and

the assays were repeated three times.

ATPase activity assay

The ATPase assay was adapted from a previously described protocol

(Chang et al, 2008). Briefly, Malachite Green reagent was prepared

by mixing 2:1:1:2 the following solutions: 0.081% malachite green,

2.3% polyvinyl alcohol, 5.7% ammonium heptamolybdate tetrahy-

drate in 6 M HCl, and H2O. 1 lM RuvbL and protein substrates at the

indicated concentrations were diluted in buffer containing 0.017%

Triton X-100, 100 mM Tris–HCl, 20 mM KCl, and 6 mM MgCl2 at

pH7.4, in the total volume of 15 ll, and dispensed into wells of

96-well white plate. The reaction started by addition of 10 ll of

2.5 mM ATP and kept at 37°C for 2 h. 80 ll of the malachite green

reagent was added to the reaction followed by 10 ll of 32% sodium

citrate to quench the reaction. After 15 min of incubation at 37°C,

the plate was read at 620 nm. Each reaction was run in triplicates.

Fibril preparation, deconstruction assay, and atomicforce microscopy

Amyloid fibrils prepared from bovine insulin (I5500, Sigma-Aldrich)

were subjected to ATPase stimulation and deconstruction assays

(Kurouski et al, 2012, 2013). Insulin was pre-incubated at 70°C and

pH 2.4 at a concentration of 60 mg/ml for 24 h to prepare fibrils.

The fibrillation process was stopped by bringing the sample to room

temperature and centrifuging twice at 16,000 g to remove the unfib-

rillated fraction. The gelatinous phase containing fibrils was then

resuspended in HCl (pH 2.4).

Samples for fibril deconstruction assays were prepared by mixing

RuvbL preparations and fibrils in a ~1:20 protein molar ratio in the

presence of 0.1 mg/ml creatine kinase, 20 mM phosphocreatine,

10 mM MgCl2, 75 mM KCl, and 20 mM HEPES buffer pH 7.4 with

or without 5 mM ATP. The reaction mixtures were incubated at

30°C for 2 h. In the control reaction, RuvbL was replaced with an

equal volume of the buffer at 30°C for 2 h followed by imaging.

Samples for atomic force microscopy (AFM) imaging were prepared

by diluting the end product of the reactions in a 1:6 volumetric ratio

by 20 mM HEPES buffer pH 7.4 and depositing on freshly cleaved

mica disks (TED PELLA, Inc., Redding, CA) for 2 min, followed by

washing with milliQ water to remove unbound materials as well as

salt, blotting, and drying under flowing air. The samples were then

imaged in tapping mode using a SmartSPMTM-1000 atomic force

microscope from AIST-NT, Inc. (Novato, CA) and gold-coated PPP-

NCHAu AFM probes (NanosensorsTM, Neuchatel, Switzerland) with

a spring constant of 42 N/m and resonance frequency of ~270 kHz.

Images were processed using the AIST-NT SPM Control Software

v.3.5.61 (AIST-NT, Inc.).

Ab fibrillation inhibition assay

Ab1–42 peptide was solubilized with 0.15 mM NaOH to final

concentration of 0.3 mM. For the experiment, the peptide was

diluted to final concentration of 10 lM with PBS buffer containing

5 mM MgCl2, 50 mM KCl, 1 mM DTT, and 20 lM thioflavin with

and without 10 mM ATP. Protein of interest mixed in the reaction,

0.1 lM RuvbL complex, or/ and HSPs (0.2 lM HSC70 and 0.2 lMDJB1). The reaction was set in 96-well black plate, transparent

bottom, and incubated at 37°C for 12 h. Fluorescence was measured

every 6 min by plate reader.

Tandem affinity purification, label-free quantitative mass spec-

trometry, chemical cross-linking mass spectrometry, yeast strains

and plasmids, yeast cultivation procedures, and thermotolerance

assays are in the Appendix Supplementary Methods.

Expanded View for this article is available online:

http://emboj.embopress.org

AcknowledgementsWe thank Daniel Kaganovich, Anindya Dutta, Erin Fenderson and I. Tsaneva for

generous gifts of strains, plasmids and reagents, and Natalie Saini for the

strain and plasmid constructions (see Appendix Supplementary Methods). This

research was supported by the US National Institutes of Health under grants

and contracts: P41 GM104603 and HHSN268201000031C (CEC), R01

GM086890 (MYS) and R01GM093294 (YOC), by the National Science Founda-

tion under awards CHE-1152752 (IKL) and MCB-0818122 (KSL), and by

Fundação para a Ciência e Tecnologia (Portugal) through the grants PTDC/

BBB-BEP/1724/2012 (STNS, TMB and PMM) and PEst-OE/EQB/LA0004/2011

(PMM). Yeast work by NVR and YOC was funded by the grant Russian Science

Foundation from 14-50-00069. The St. Petersburg State University support

from projects 1.50.2218.2013 (YOC) and IAS 1.42.1284.2014 (travel support to

NVR) contributed to the preparation of this paper.

Author contributionsNZ performed the main body of experiments, designed experiments, and

analyzed data. PL and PMM provided purified proteins and technical expertise.

XX and MM performed experiments with mass spectrometry. CEC provided

expertise in mass spectrometry. ABM performed experiments with cell culture.

GPN, RG, and NVR performed experiments with yeast. MS performed experi-

ments with atomic force microscopy. IKL provided technical expertise in atomic

force microscopy. STNS and TMB purified proteins. KSL directed constructions

of strains and plasmids. YOC and MYS designed research and analyzed data.

Conflict of interestThe authors declare that they have no conflict of interest.

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RuvbL1 and RuvbL2 enhance aggresome formation and...

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