Candida albicans ESCRT Pathway Makes Rim101-Dependent … · candidiasis, found that the vps27/...

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EUKARYOTIC CELL, Aug. 2010, p. 1203–1215 Vol. 9, No. 8 1535-9778/10/$12.00 doi:10.1128/EC.00056-10 Copyright © 2010, American Society for Microbiology. All Rights Reserved. The Candida albicans ESCRT Pathway Makes Rim101-Dependent and -Independent Contributions to Pathogenesis Julie M. Wolf, Diedre J. Johnson, David Chmielewski, and Dana A. Davis* Department of Microbiology, University of Minnesota, Minneapolis, Minnesota 55110 Received 3 March 2010/Accepted 15 June 2010 Candida albicans is an opportunistic pathogen that colonizes diverse mucosal niches with distinct environ- mental characteristics. To adapt to these different sites, C. albicans must activate and attenuate a variety of signal transduction pathways. A mechanism of signal attenuation is through receptor endocytosis and subse- quent vacuolar degradation, which requires the endosomal sorting complex required for transport (ESCRT) pathway. This pathway comprises several polyprotein complexes (ESCRT-0, -I, -II, -III, and -DS) that are sequentially recruited to the endosomal membrane. The ESCRT pathway also activates the Rim101 transcription factor, which governs expression of genes required for virulence. Here, we tested the hypothesis that the ESCRT pathway plays a Rim101-independent role(s) in pathogenesis. We generated deletion mutants in each ESCRT complex and determined that ESCRT-I, -II, and -III are required for Rim101 activation but that ESCRT-0 and ESCRT-DS are not. We found that the ESCRT-0 member Vps27 and ESCRT-DS components are required to promote epithelial cell damage and, using a murine model of oral candidiasis, found that the vps27/ mutant had a decreased fungal burden compared to that of the wild type. We found that a high-dose inoculum can compensate for fungal burden defects but that mice colonized with the vps27/ strain exhibit less morbidity than do mice infected with the wild-type strain. These results demonstrate that the ESCRT pathway has Rim101-independent functions for C. albicans virulence. Candida albicans is a ubiquitous human commensal that can cause infections in susceptible hosts (33, 41). In both disease and nondisease states, C. albicans colonizes diverse mucosal surfaces of its host, including the oral cavity, gastrointestinal tract, and urogenital tract (32). These niches can vary widely in pH, osmolarity, and available nutrients. C. albicans therefore survives in diverse environmental niches within its human host. To adapt to these specific niches, C. albicans must be able to activate specific signal transduction pathways and concomi- tantly terminate other pathways to properly respond to envi- ronmental cues and regulate gene expression. One mechanism to terminate a signaling pathway is to degrade the cognate receptors in the vacuole. Thus, niche adaptation requires tight regulation of signaling pathway activation and termination, and receptor degradation is one mechanism to terminate signal propagation. Prior to vacuolar degradation, the receptor protein is inter- nalized through endocytosis, where it is subsequently incorpo- rated into the endosomal lumen as an interluminal vesicle (ILV). Formation of the ILV requires the endosomal sorting pathway required for transport (ESCRT) complex pathway (3, 39, 42). ILVs increase in number, generating a mature mul- tivesicular body (MVB), which then fuses with and releases its luminal content into the vacuole for degradation. MVB for- mation allows for transmembrane receptor downregulation and contributes to signal transduction arrest during adaptation to new environments. The ESCRT complex is composed of heterogeneous polypro- tein complexes recruited to the endosomal membrane by the posttranslational modification pattern, frequently ubiquitination, of the receptor (or cargo) protein and the membrane lipid con- tent (22). There are four core polyprotein complexes (Fig. 1): ESCRT-0 (Vps27 and Hse1), ESCRT-I (Mvb12, Vps23, Vps37, and Vps28), ESCRT-II (Vps36, Vps22, and Vps25), and ESCRT-III (Vps20, Snf7, Vps2, and Vps24) (2, 3, 7, 13, 21, 22). These complexes are recruited sequentially, and while ESCRT-0, -I, and -II are recruited as fully formed complexes, ESCRT-III arrives as two separate heterodimers (2). The first heterodimer, Vps20-Snf7, facilitates nucleation of additional Snf7 monomers around the ubiquitinated cargo protein (42), and Snf7 oligomer- ization is thought to mediate membrane involution necessary for ILV formation (16, 40, 42). The second ESCRT-III heterodimer, Vps2-Vps24, caps the Snf7 oligomer (2, 42) and recruits down- stream proteins such as Bro1, Doa4, and Vps4 (4, 30). Bro1 recruits Doa4, which deubiquitinates the cargo protein before its incorporation into the ILV. Vps4, an AAA-ATPase, catalyzes ESCRT-III dissociation from the membrane and formation of the ILV (3, 4). The ESCRT complex function thereby results in ILV and subsequent MVB formation. In addition to forming MVBs, the ESCRT-III member Snf7 plays a direct role in signal transduction in C. albicans and other fungi (24, 47, 49). Snf7 interacts with Rim20 (9, 20), a scaffold protein that binds the Rim101 transcription factor (48), and with Rim13 (8, 20), the putative protease responsible for Rim101 proteolytic activation (Fig. 1) (25). Activation of Rim101 is required for adaptation to neutral-alkaline environ- ments (15), and this activation is mediated by Snf7 recruitment of Rim13 and Rim20. Snf7 is therefore required for both MVB formation and Rim101 processing in C. albicans. Snf7 localization to the endocytic membrane is important for * Corresponding author. Mailing address: Department of Microbi- ology, University of Minnesota, 1360 Mayo Building MMC196, 420 Delaware Street SE, Minneapolis, MN 55455. Phone: (612) 624-1912. Fax: (612) 626-0623. E-mail: [email protected]. Published ahead of print on 25 June 2010. 1203 on April 12, 2019 by guest http://ec.asm.org/ Downloaded from

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EUKARYOTIC CELL, Aug. 2010, p. 1203–1215 Vol. 9, No. 81535-9778/10/$12.00 doi:10.1128/EC.00056-10Copyright © 2010, American Society for Microbiology. All Rights Reserved.

The Candida albicans ESCRT Pathway Makes Rim101-Dependentand -Independent Contributions to Pathogenesis�

Julie M. Wolf, Diedre J. Johnson, David Chmielewski, and Dana A. Davis*Department of Microbiology, University of Minnesota, Minneapolis, Minnesota 55110

Received 3 March 2010/Accepted 15 June 2010

Candida albicans is an opportunistic pathogen that colonizes diverse mucosal niches with distinct environ-mental characteristics. To adapt to these different sites, C. albicans must activate and attenuate a variety ofsignal transduction pathways. A mechanism of signal attenuation is through receptor endocytosis and subse-quent vacuolar degradation, which requires the endosomal sorting complex required for transport (ESCRT)pathway. This pathway comprises several polyprotein complexes (ESCRT-0, -I, -II, -III, and -DS) that aresequentially recruited to the endosomal membrane. The ESCRT pathway also activates the Rim101transcription factor, which governs expression of genes required for virulence. Here, we tested thehypothesis that the ESCRT pathway plays a Rim101-independent role(s) in pathogenesis. We generateddeletion mutants in each ESCRT complex and determined that ESCRT-I, -II, and -III are required forRim101 activation but that ESCRT-0 and ESCRT-DS are not. We found that the ESCRT-0 member Vps27and ESCRT-DS components are required to promote epithelial cell damage and, using a murine model of oralcandidiasis, found that the vps27�/� mutant had a decreased fungal burden compared to that of the wild type.We found that a high-dose inoculum can compensate for fungal burden defects but that mice colonized withthe vps27�/� strain exhibit less morbidity than do mice infected with the wild-type strain. These resultsdemonstrate that the ESCRT pathway has Rim101-independent functions for C. albicans virulence.

Candida albicans is a ubiquitous human commensal that cancause infections in susceptible hosts (33, 41). In both diseaseand nondisease states, C. albicans colonizes diverse mucosalsurfaces of its host, including the oral cavity, gastrointestinaltract, and urogenital tract (32). These niches can vary widely inpH, osmolarity, and available nutrients. C. albicans thereforesurvives in diverse environmental niches within its human host.

To adapt to these specific niches, C. albicans must be able toactivate specific signal transduction pathways and concomi-tantly terminate other pathways to properly respond to envi-ronmental cues and regulate gene expression. One mechanismto terminate a signaling pathway is to degrade the cognatereceptors in the vacuole. Thus, niche adaptation requires tightregulation of signaling pathway activation and termination,and receptor degradation is one mechanism to terminate signalpropagation.

Prior to vacuolar degradation, the receptor protein is inter-nalized through endocytosis, where it is subsequently incorpo-rated into the endosomal lumen as an interluminal vesicle(ILV). Formation of the ILV requires the endosomal sortingpathway required for transport (ESCRT) complex pathway (3,39, 42). ILVs increase in number, generating a mature mul-tivesicular body (MVB), which then fuses with and releases itsluminal content into the vacuole for degradation. MVB for-mation allows for transmembrane receptor downregulationand contributes to signal transduction arrest during adaptationto new environments.

The ESCRT complex is composed of heterogeneous polypro-tein complexes recruited to the endosomal membrane by theposttranslational modification pattern, frequently ubiquitination,of the receptor (or cargo) protein and the membrane lipid con-tent (22). There are four core polyprotein complexes (Fig. 1):ESCRT-0 (Vps27 and Hse1), ESCRT-I (Mvb12, Vps23, Vps37,and Vps28), ESCRT-II (Vps36, Vps22, and Vps25), andESCRT-III (Vps20, Snf7, Vps2, and Vps24) (2, 3, 7, 13, 21, 22).These complexes are recruited sequentially, and while ESCRT-0,-I, and -II are recruited as fully formed complexes, ESCRT-IIIarrives as two separate heterodimers (2). The first heterodimer,Vps20-Snf7, facilitates nucleation of additional Snf7 monomersaround the ubiquitinated cargo protein (42), and Snf7 oligomer-ization is thought to mediate membrane involution necessary forILV formation (16, 40, 42). The second ESCRT-III heterodimer,Vps2-Vps24, caps the Snf7 oligomer (2, 42) and recruits down-stream proteins such as Bro1, Doa4, and Vps4 (4, 30). Bro1recruits Doa4, which deubiquitinates the cargo protein before itsincorporation into the ILV. Vps4, an AAA-ATPase, catalyzesESCRT-III dissociation from the membrane and formation of theILV (3, 4). The ESCRT complex function thereby results in ILVand subsequent MVB formation.

In addition to forming MVBs, the ESCRT-III member Snf7plays a direct role in signal transduction in C. albicans andother fungi (24, 47, 49). Snf7 interacts with Rim20 (9, 20), ascaffold protein that binds the Rim101 transcription factor(48), and with Rim13 (8, 20), the putative protease responsiblefor Rim101 proteolytic activation (Fig. 1) (25). Activation ofRim101 is required for adaptation to neutral-alkaline environ-ments (15), and this activation is mediated by Snf7 recruitmentof Rim13 and Rim20. Snf7 is therefore required for both MVBformation and Rim101 processing in C. albicans.

Snf7 localization to the endocytic membrane is important for

* Corresponding author. Mailing address: Department of Microbi-ology, University of Minnesota, 1360 Mayo Building MMC196, 420Delaware Street SE, Minneapolis, MN 55455. Phone: (612) 624-1912.Fax: (612) 626-0623. E-mail: [email protected].

� Published ahead of print on 25 June 2010.

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both MVB and Rim101 functions of Snf7 and is dependent onthe upstream ESCRT complex members. In Saccharomycescerevisiae, mutations in ESCRT-I or -II or the first heterodimerof ESCRT-III prevent Snf7 recruitment to the endosomalmembrane and the ability of these strains to form MVBs.These mutants are also unable to process Rim101 (38, 49).ESCRT component mutations downstream of SNF7 preventnormal MVB formation but not Rim101 processing (38, 49).Likewise, mutation of the ESCRT-0 component VPS27 dis-rupts MVB formation but not Rim101 processing in S. cerevi-siae. These data demonstrate that ESCRT-0 and ESCRT-DSare not required for Rim101 processing in S. cerevisiae but thatESCRT-I and -II and the first ESCRT-III heterodimer are (38,49). This suggests that Snf7 must be recruited to the endosomefor either MVB-specific or Rim101-specific function. Snf7 andRim20 exhibit alkaline pH-dependent colocalization in a punc-tate pattern reminiscent of endosomal staining (10), suggestingthat extracellular environment can influence Snf7 interactions.Snf7 therefore acts as a molecular hub at the endosome thatcan coordinate MVB-specific or Rim101-specific functions.

The role of ESCRT components in C. albicans Rim101processing is relatively unexplored. Mutants in C. albicansESCRT-I, -II, and -IIIa components displayed filamentationdefects when tested for Rim101-dependent filamentation (49),suggesting that these genes are required for Rim101-depen-dent phenotypes. However, no ESCRT-0 C. albicans mutantshave been tested for filamentation or Rim101 processing.Thus, the critical ESCRT complexes for C. albicans Rim101processing are not fully understood.

We wished to investigate the relationship between theESCRT and Rim101 pathways in C. albicans and to determinewhether C. albicans pathogenesis requires ESCRT functionindependent of Rim101. Previous studies have suggested thatESCRT function plays an independent role in bloodstreamcandidiasis by demonstrating that mice infected with vps28�/�

or vps32�/� C. albicans strains succumb to infection moreslowly than do mice infected with a rim101�/� C. albicansstrain (11). However, these mutant strains have not fullydecoupled the Rim101 and ESCRT pathways, as these mu-tations affect both pathways. We hypothesized that, like S.cerevisiae, ESCRT-0 would not be required for Rim101 pro-cessing and would therefore be an ideal candidate in whichto study MVB-specific contributions of the ESCRT pathway.Here, we generated a collection of ESCRT component mu-tants to characterize Rim101- and ESCRT-dependent phe-notypes. We then used these mutants to investigate the roleof ESCRT function in epithelial cell damage and C. albicansvirulence. Our results demonstrate a role for ESCRT com-plex function in C. albicans pathogenesis independent ofeffects on Rim101 activation.

MATERIALS AND METHODS

Media and growth conditions. All C. albicans strains were routinely passagedin YPD (1% yeast extract, 2% [wt/vol] Bacto peptone, 2% [wt/vol] dextrose)supplemented with 80 �g/ml uridine (Sigma). To select for Arg�, Ura�, or His�

transformants, synthetic medium (0.67% yeast nitrogen base plus ammoniumsulfate and without amino acids; 2% dextrose; 80 �g of uridine per ml) was used,supplemented as required by the auxotrophic needs of the cells (1). To test forRim101-dependent growth phenotypes, YPD was buffered with 150 mM HEPESto pH 9 with NaOH or contained 150 mM LiCl. For alkaline-induced filamen-tation assays, M199 medium (Gibco BRL) was buffered with 150 mM HEPESand pH adjusted to pH 4 or pH 8 and supplemented with 80 �g/ml uridine. Forserum-induced filamentation assays, 4% bovine calf serum (BCS) was supple-mented with 80 �g/ml uridine. For determining CFU from mouse tissue, YPDwas supplemented with 1 �g/ml ampicillin (Invitrogen). Solid medium was pre-pared as described above with the addition of 2% Bacto agar.

Strain manipulation. All strain manipulations were performed in the BWP17genetic background and are listed in Table 1. All knockouts were engineered bysubsequent rounds of allelic exchange with PCRs using a set of disrupt (DR)primers specific for the targeted gene (Table 2). For example, to generate thevps27�/� strain (DAY1177), the vps27::ARG4 cassette was amplified in a PCRusing Vps27 5DR and Vps27 3DR primers and pRS-ARG�Spe (46) template togenerate the heterozygous mutant DAY1176. DAY1176 was transformed withthe vps27::URA3-dpl200 cassette, which was amplified in a PCR using Vps275DR and Vps27 3DR primers and pDDB57 template (45), to generate thehomozygous mutant DAY1177. To generate a prototrophic His� strain,DAY1177 was transformed with NruI-digested pDDB78 plasmid to generateDAY1160. For the VPS27 complementation vector, genomic VPS27 plus pro-moter and terminator sequence was amplified in a PCR using Vps27 5comp andVps27 3comp primers and DAY1 genomic DNA as a template. This sequencewas transformed into S. cerevisiae along with NotI/EcoRI-digested pDDB78 togenerate pDDB501 by in vivo recombination (28). NruI-digested pDDB501 wastransformed into DAY1177 to generate DAY1264. All allelic exchange resultswere confirmed by diagnostic PCR (Fig. 2 and data not shown). To expressRIM101-V5 in the vps27�/� strain, NruI-digested pDDB233 (25) was trans-formed into DAY1177 to generate DAY1178. All other ESCRT mutants weremade similarly (25).

Growth assays. Overnight YPD cultures were diluted 1:20 in sterile water anddiluted subsequently 1:5 into sterile water using a 96-well sterile plate. Fivemicroliters of each dilution was spotted onto YPD, YPD plus LiCl, and YPD pH9 plates and incubated for 2 days at 37°C. For growth assays under iron-limitingconditions, strains were grown in YPD containing 50 �M iron chelator batho-phenanthrolinedisulfonic acid (BPS) for 2 days before being spotted in 5-foldserial dilutions onto YPD plus 150 �M BPS plates. Plates were photographedwith a Canon Powershot A560 camera and adjusted in Adobe Photoshop Ele-ments 2.0.

Filamentation assays. Strains were grown in 3 ml YPD overnight at 30°C. Foralkaline-induced filamentation assays, 3-�l overnight cultures were spotted ontoM199 pH 8 agar and incubated for 5 days at 37°C. For serum-induced filamen-tation, 3-�l overnight cultures were spread on 4% BCS plates and incubated for24 h at 37°C. Strains were visualized and assessed for filamentation on a ZeissImager.M1 microscope.

Protein preparations. Strains were grown to mid-log phase in 40 ml M199 pH4 medium and either collected directly for protein preparation or collected and

FIG. 1. Model of ESCRT pathway and Rim101 pathway inter-section. The Rim101 pathway intersects with the ESCRT pathway.ESCRT-0, -I, -II, and -III complexes are recruited to the endosomalmembrane. ESCRT-III member Snf7 can interact either with Rim101pathway members, resulting in proteolytic activation of Rim101, orwith downstream ESCRT members, resulting in MVB formation.Yeast ESCRT complex components are listed below, and designationsin bold are of those investigated in this study.

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resuspended in 40 ml M199 pH 8 for 30 min before collection. Cells werecollected by centrifugation and washed in 1 mM phenylmethylsulfonyl fluoride(PMSF), and cell pellets were resuspended in radioimmunoprecipitation assaybuffer (50 mM Tris [pH 8], 150 mM NaCl, 1% NP-40, 3 mM EDTA, 0.5%deoxycholate, 0.1% sodium laurel sulfate) with protease inhibitors (1 mM PMSFand 1 �g/ml each of aprotinin, pepstatin, and leupeptin) and 10 mM dithiothre-itol. Cells were lysed by glass bead disruption by being vortexed four times at 4°C.Lysates were then cleared by centrifugation at 15,000 � g for 15 min at 4°C toremove cell debris.

Western blot analysis. A 50-�l protein preparation was separated by SDS-polyacrylamide gel electrophoresis (PAGE) in an 8% resolving gel. Gels weretransferred to a nitrocellulose membrane and blocked for 1 h at room temper-ature with 5% dry milk dissolved in Tris-buffered saline containing 0.05% Tween20 (TBS-T). Membranes were incubated with blocking solution containing1:5,000 monoclonal anti-V5-horseradish peroxidase (HRP) conjugate antibody(Invitrogen). Membranes were washed three times in TBS-T, incubated withECL reagent (Amersham), and exposed to film.

FaDu cell damage assay. FaDu cells (ATCC) were plated in 24-well tissueculture dishes and incubated at 37°C and 5% CO2 in modified Eagle medium(MEM) with a 10% final concentration of fetal bovine serum (FBS) and 5 mlantibiotic/antimycotic cocktail (Invitrogen). At 90% monolayer confluence, cellswere incubated in 0.5 ml medium containing 0.5 �Ci Cr51 for 16 h. After theFaDu cells were washed with phosphate-buffered saline (PBS), 1 � 105 C.albicans cells were added in MEM with 10% FBS and 5 ml antibiotic cocktail(Invitrogen) and incubated for 10 h. Some FaDu cells were left uninfected tomeasure spontaneous Cr51 release. An 0.5-ml amount of supernatant was movedto a 13-ml glass test tube. An 0.5-ml amount of 6 M NaOH was added to theFaDu cells, the entire volume was moved to a separate test tube, and a final washwith 0.5 ml Liftaway (RPI Corp.) was performed. Specific release was calculated

as [(2 � supernatant) � (2 � spontaneous release)]/[(2 � total) � (2 � spon-taneous release)]. All samples were run in triplicate.

Oropharyngeal candidiasis (OPC) mouse model. BALB/c mice 4 to 6 weeksold were obtained from Charles River (Wilmington, MA). On day �1, mice wereimmunosuppressed with 0.2 mg cortisone acetate/g of body weight. On day 0, 50�l of 1 � 105-cell/ml or 1 � 106-cell/ml C. albicans cell suspensions was inocu-lated onto a sterilized cotton ball and incubated for 1 h in the oral cavity of miceanesthetized with 100 �g/g ketamine, 20 �g/g xylazine-HCl, and 2.8 �g/gacepromazine. On day 3, 10 mice per C. albicans strain were sacrificed, and thetongues were removed, weighed, and saved in 2 ml PBS. Tissues were homoge-nized and plated in 100, 10�1, and 10�2 dilutions on YPD plus ampicillin (AMP)plates. Plates were incubated at 37°C overnight. Colonies were counted andcalculated to represent CFU/g tissue. On day 3, remaining mice were reimmu-nosuppressed with 0.2 mg/g cortisone acetate. On day 6, the remaining mice weresacrificed and tongues were homogenized and plated as described for day 3. Allmice were weighed daily throughout the course of infection. All experimentswere performed under the established University of Minnesota InstitutionalAnimal Care and Use Committee guidelines.

RESULTS

ESCRT knockout mutations and ESCRT-dependent pheno-typic defects. We hypothesized that MVB formation plays arole in C. albicans pathogenesis independent of Rim101processing. To test this hypothesis, we generated and char-acterized strains with mutations in individual members ofthe ESCRT-0 (Vps27 and Hse1), ESCRT-I (Mvb12 and

TABLE 1. Strains used in these studies

Name Genotype Reference

BWP17 ura3::�imm434/ura3::�imm434 arg4::hisG/arg4::hisG his1::hisG/his1::hisG 46DAY25 ura3::�imm434/ura3::�imm434 arg4::hisG/arg4::hisG HIS1::DDB78::his1::hisG/his1::hisG rim101::ARG4/rim101::URA3 15DAY185 ura3::�imm434/ura3::�imm434 ARG4::URA3::arg4::hisG/arg4::hisG HIS1::DDB78::his1::hisG/his1::hisG 14DAY534 ura3::�imm434/ura3::�imm434 arg4::hisG/arg4::hisG his1::hisG/his1::hisG snf7::ARG4/snf7::URA3-dpl200 24DAY537 ura3::�imm434/ura3::�imm434 arg4::hisG/arg4::hisG his1::hisG/his1::hisG vps4::ARG4/vps4::URA3-dpl200 24DAY568 ura3::�imm434/ura3::�imm434 arg4::hisG/arg4::hisG RIM101-V5::HIS1::DDB233:::his1::hisG/his1::hisG vps4::ARG4/vps4::URA3-dpl200 24DAY576 ura3::�imm434/ura3::�imm434 arg4::hisG/arg4::hisG RIM101-V5::HIS1::DDB233:::his1::hisG/his1::hisG vps4::ARG4 vps4::URA3-dpl200 24DAY653 ura3::�imm434/ura3::�imm434 arg4::hisG/arg4::hisG his1::hisG/his1::hisG bro1::ARG4/bro1::URA3-dpl200 24DAY763 ura3::�imm434/ura3::�imm434 arg4::hisG/arg4::hisG HIS1::DDB78::his1::hisG/his1::hisG snf7::ARG4/snf7::URA3-dpl200 24DAY1155 ura3::�imm434/ura3::�imm434 arg4::hisG/arg4::hisG HIS1::DDB78::his1::hisG/his1::hisG vps4::ARG4/vps4::URA3-dpl200 This studyDAY1156 ura3::�imm434/ura3::�imm434 arg4::hisG/arg4::hisG HIS1::DDB78::his1::hisG/his1::hisG bro1::ARG4/bro1::URA3-dpl200 This studyDAY1157 ura3::�imm434/ura3::�imm434 arg4::hisG/arg4::hisG HIS1::DDB78::his1::hisG/his1::hisG vps20::ARG4/vps20::URA3-dpl200 This studyDAY1158 ura3::�imm434/ura3::�imm434 arg4::hisG/arg4::hisG HIS1::DDB78::his1::hisG/his1::hisG doa4::ARG4/doa4::URA3-dpl200 This studyDAY1159 ura3::�imm434/ura3::�imm434 arg4::hisG/arg4::hisG HIS1::DDB78::his1::hisG/his1::hisG bro1::ARG4/bro1::URA3-dpl200 This studyDAY1160 ura3::�imm434/ura3::�imm434 arg4::hisG/arg4::hisG HIS1::DDB78::his1::hisG/his1::hisG vps27::ARG4/vps27::URA3-dpl200 This studyDAY1161 ura3::�imm434/ura3::�imm434 arg4::hisG/arg4::hisG HIS1::DDB78::his1::hisG/his1::hisG vps28::ARG4/vps28::URA3-dpl200 This studyDAY1162 ura3::�imm434/ura3::�imm434 arg4::hisG/arg4::hisG HIS1::DDB78::his1::hisG/his1::hisG vps36::ARG4/vps36::URA3-dpl200 This studyDAY1163 ura3::�imm434/ura3::�imm434 arg4::hisG/arg4::hisG HIS1::DDB78::his1::hisG/his1::hisG mvb12::ARG4/mvb12::URA3-dpl200 This studyDAY1176 ura3::�imm434/ura3::�imm434 arg4::hisG/arg4::hisG his1::hisG/his1::hisG vps27::ARG4/VPS27 This studyDAY1177 ura3::�imm434/ura3::�imm434 arg4::hisG/arg4::hisG his1::hisG/his1::hisG vps27::ARG4/vps27::URA3-dpl200 This studyDAY1178 ura3::�imm434/ura3::�imm434 arg4::hisG/arg4::hisG RIM101-V5::DDB233::his1::hisG/his1::hisG vps27::ARG4/vps27::URA3-dpl200 This studyDAY1181 ura3::�imm434/ura3::�imm434 arg4::hisG/arg4::hisG his1::hisG/his1::hisG vps28::ARG4/VPS28 This studyDAY1182 ura3::�imm434/ura3::�imm434 arg4::hisG/arg4::hisG his1::hisG/his1::hisG vps28::ARG4/vps28::URA3-dpl200 This studyDAY1183 ura3::�imm434/ura3::�imm434 arg4::hisG/arg4::hisG RIM101-V5::DDB233::his1::hisG/his1::hisG vps28::ARG4/vps28::URA3-dpl200 This studyDAY1186 ura3::�imm434/ura3::�imm434 arg4::hisG/arg4::hisG his1::hisG/his1::hisG vps36::ARG4/VPS36 This studyDAY1187 ura3::�imm434/ura3::�imm434 arg4::hisG/arg4::hisG his1::hisG/his1::hisG vps36::ARG4/vps36::URA3-dpl200 This studyDAY1188 ura3::�imm434/ura3::�imm434 arg4::hisG/arg4::hisG RIM101-V5::DDB233::his1::hisG/his1::hisG vps36::ARG4/vps36::URA3-dpl200 This studyDAY1191 ura3::�imm434/ura3::�imm434 arg4::hisG/arg4::hisG his1::hisG/his1::hisG mvb12::ARG4/MVB12 This studyDAY1192 ura3::�imm434/ura3::�imm434 arg4::hisG/arg4::hisG his1::hisG/his1::hisG mvb12::ARG4/mvb12::URA3-dpl200 This studyDAY1193 ura3::�imm434/ura3::�imm434 arg4::hisG/arg4::hisG RIM101-V5::DDB233::his1::hisG/his1::hisG mvb12::ARG4/mvb12::URA3-dpl200 This studyDAY1196 ura3::�imm434/ura3::�imm434 arg4::hisG/arg4::hisG his1::hisG/his1::hisG vps22::ARG4/VPS22 This studyDAY1197 ura3::�imm434/ura3::�imm434 arg4::hisG/arg4::hisG his1::hisG/his1::hisG vps22::ARG4/vps22::URA3-dpl200 This studyDAY1200 ura3::�imm434/ura3::�imm434 arg4::hisG/arg4::hisG his1::hisG/his1::hisG hse1::ARG4/HSE1 This studyDAY1201 ura3::�imm434/ura3::�imm434 arg4::hisG/arg4::hisG his1::hisG/his1::hisG hse1::ARG4/hse1::URA3-dpl200 This studyDAY1212 ura3::�imm434/ura3::�imm434 arg4::hisG/arg4::hisG RIM101-V5::DDB233::his1::hisG/his1::hisG rim101::ARG4/rim101::URA3-dpl200 This studyDAY1217 ura3::�imm434/ura3::�imm434 arg4::hisG/arg4::hisG HIS1::DDB78::his1::hisG/his1::hisG vps22::ARG4/vps22::URA3-dpl200 This studyDAY1218 ura3::�imm434/ura3::�imm434 arg4::hisG/arg4::hisG HIS1::DDB78::his1::hisG/his1::hisG hse1::ARG4/hse1::URA3-dpl200 This studyDAY1219 ura3::�imm434/ura3::�imm434 arg4::hisG/arg4::hisG RIM101-V5::DDB233::his1::hisG/his1::hisG bro1::ARG4/bro1::URA3-dpl200 This studyDAY1221 ura3::�imm434/ura3::�imm434 arg4::hisG/arg4::hisG RIM101-V5::DDB233::his1::hisG/his1::hisG hse1::ARG4/hse1::URA3-dpl200 This studyDAY1222 ura3::�imm434/ura3::�imm434 arg4::hisG/arg4::hisG RIM101-V5::DDB233::his1::hisG/his1::hisG vps22::ARG4/vps22::URA3-dpl200 This studyDAY1264 ura3::�imm434/ura3::�imm434 arg4::hisG/arg4::hisG VPS27::HIS1::DDB501::his1::hisG/his1::hisG vps27::ARG4/vps27::URA3-dpl200 This studyDAY750

(CT128.1)ura3::�imm434/ura3::�imm434 arg4::hisG/arg4::hisG his1::hisG/his1::hisG ftr1::ARG4/ftr1::URA3-dpl200 23

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Vps28), ESCRT-II (Vps36 and Vps22), or ESCRT-III (Vps20and Snf7) complexes, as well as mutations in components thatact downstream of Snf7 (Vps4, Bro1, and Doa4) (referred to asESCRT-DS in this text) (Fig. 1 and Table 1). All mutant strainswere made prototrophic and were then tested for ESCRT-dependent and Rim101-dependent phenotypes.

We first tested whether ESCRT homologs in C. albicansfunction in endosomal trafficking using the lipophilic dye FM4-64 to visualize internalized vesicle trafficking (43). Theplasma membrane was stained with FM 4-64, and the unbounddye was removed by washing. Following a 60-min chase, wild-type cells showed strong staining of the vacuole, with little tono cytoplasmic staining (Fig. 3A). Although the mvb12�/�,vps4�/�, bro1�/�, and doa4�/� strains displayed weaker de-fects than did the other ESCRT mutants tested, all strainslacking ESCRT components displayed staining patterns con-taining accumulations of perivacuolar staining, indicating thateach of the individual mutations in ESCRT complex genesleads to formation of class E-like exclusion bodies (Fig. 3A). In

S. cerevisiae, most ESCRT components were initially identifiedas class E vps mutants, due to the formation of a “class Eexclusion body” around the vacuole perimeter, which consistsof accumulated immature and/or incomplete MVBs unable tofuse with the vacuole (36). This demonstrates that the ho-mologs identified in C. albicans play a similar role in endoso-mal trafficking as they do in other organisms. We concludedthat all ESCRT components identified play a role in MVBtrafficking.

Rim101-dependent phenotypic defects. We next determinedthe effect of the ESCRT mutants on Rim101-dependent pro-cesses in C. albicans. Overnight YPD cultures were diluted1:50 in PBS, and 5-fold dilutions were spotted onto YPD, YPDpH 9, or YPD plus LiCl agar medium. After 2 days at 37°C, allstrains grew robustly on YPD, with similar colony formationnumbers at higher dilutions, indicating that each dilution setrepresented roughly equivalent numbers of cells (Fig. 4). Thus,all mutants were able to grow well on rich medium and at aphysiologically relevant temperature.

As expected, we observed robust growth of the wild-typestrain and poor growth of the rim101�/� strain on YPD pH 9and YPD plus LiCl (Fig. 4). However, the vps27�/� and thehse1�/� strains, which lack individual ESCRT-0 components,grew similarly to wild type on both YPD pH 9 and YPD plusLiCl. Thus, ESCRT-0 is not required for Rim101-dependentgrowth.

As predicted, all strains lacking ESCRT-I (mvb12�/� orvps28�/�), ESCRT-II (vps36�/� or vps22�/�), or either com-ponent of the Vps20-Snf7 ESCRT-III heterodimer grew poorlyon both YPD pH 9 and YPD plus LiCl (Fig. 4). This extendsbut is consistent with previously reported data from S. cerevi-

FIG. 2. PCR genotyping of vps27�/� strains. VPS27 was amplifiedin a PCR using Vps27 5det and Vps27 3det primers (Table 2). Sizemarkers in kilobases are noted on the left, and genotypes of each bandare labeled on the right. Strains shown are VPS27/VPS27 (DAY185),VPS27/� (DAY1176), vps27�/� (DAY1160), and vps27�/� � VPS27(DAY1264) strains. WT, wild type.

TABLE 2. Primers used in these studies

Primer name Primer sequence

Vps27 5DR .............................AATACTTATTAATTTCACATATAATATCATTTCATTAGTAGTAGGAAATACCTTTATATTTTTCCCAGTCACGACGTTVps27 3DR .............................AGAAGTAGAAGTTTGGCTAAATATTTCTAGATAAGCAAAAATTATTGCCAACTTTTATTAGTGGAATTGTGAGCGGATAHse1 5DR ...............................TTTGCCTCCTCCCACTATTAATCAATTAGTAACCCCGATCATCAATTCCTTACCAACAACCTTTCCCAGTCACGACGTTHse1 3DR ...............................AGTACTACAGCTTTATACTTCTTTCAATAGAAGAATATTAGTGAGACGTGTATTATATGACTGGAATTGTGAGCGGATAMvb12 5DR ............................GCAACCAACGAACGAACGAACGAACGAGCGAACCTATAAACACATACATTCACAACAACATTTCCCAGTCACGACGTTMvb12 3DR ............................TTATACTATTAATAATATGACCCATCTATTTACAAAAAAAAAACGTTTCATTATAATTCAGTGGAATTGTGAGCGGATAVps28 5DR .............................TTTCTTTAGTGTTTTGTTTATCCTTAGTCATAGAACAATCAACTTTGATTTATTGCCATTTTTCCCAGTCACGACGTTVps28 3DR .............................ATCGTATAAGCAAGAAACAGAGTATCCAACCAAACAGATAATTGTGTATTGTGTATATTAGTGGAATTGTGAGCGGATAVps36 5DR .............................TAGATGGGGAGTGGGAGCGAGTCAGATCTAATCTCTTATATATAGAAAAACCTCAATATTTTTCCCAGTCACGACGTTVps36 3DR .............................ATTAATACATAGTTCTATATATATGTCCATTTTTTTTTTTAAAAAAAACATATTACTTTAGTGGAATTGTGAGCGGATAVps22 5DR .............................ATTTTGATTAAAAGAGAAATCAAATCTTTATCTATCTTCCTATCACTTAACTATACTATTTTTCCCAGTCACGACGTTVps22 3DR .............................GGACAAGAAAAACTCAATAAAGAATAAGAAGTAAGTATGCCTGTATATATTTATTATTTAGTGGAATTGTGAGCGGATAVps20 5DR .............................TTATCAGGAACTGTAATTTGCTATCATAATCAATATAGATCTAATACATTCAAATTGACATTTCCCAGTCACGACGTTVps20 3DR .............................ATGTAAATGCAAAAATTTATTATTAAGTGTATTATGACTCACATAGAATTATGTTGTTGGGTGGAATTGTGAGCGGATADoa4 5DR...............................TTCGTACACTTTCTGATTCCAAACTAAATTACCACCAACTCTAACTTTTGTTCTTTGAAGTTTCCCAGTCACGACGTTDoa4 3DR...............................GGGACACCACCCCAAGTTTCAATTTATTGACAAAAATAAATCTATGAGTTCTATTTAATAGTGGAATTGTGAGCGGATAVps27 5det ..............................CCAGACGATATCAAACCTCCVps27 3det ..............................CGAAAAAGATTAACACTACGHse1 5det ................................CCTCTGCTTATTCCAATTAGAACCHse1 3det ................................CGGTCAAGAAATCAAGGCTGACCCMvb12 5det .............................TCTCCAATCAAAGTGTTGTCMvb12 3det .............................GGTGTTCATCTTCCGATTAGVps28 5det ..............................CGTCTGTGAATCTCATGGTGVps28 3det ..............................CTCAAAGTTAGCATCGGACAVps36 5det ..............................GACAAGAATAAGACCACGAGVps36 3det ..............................CTCAACGTTATTTTTCTTCVps22 5det ..............................GGCTCTGTATGGTCAATGAATAGCVps22 3det ..............................CCCATCAATGGAATGGAAATCCVps20 5det ..............................GAGGGGGATCAAGTTGCAAAVps20 3det ..............................CCTTCCTGATTTTGCAAGACDoa4 5det ...............................AAAATAAAGGAAATCCTGCADoa4 3det ...............................ACGGCGAAATACACATAAATVps27 5comp ..........................AAGCTCGGAATTAACCCTCACTAAAGGGAACAAAAGCTGGCCTCTTCTTATCCAGTTTACTGVps27 3comp ..........................ACGACGGCCAGTGAATTGTAATACGACTCACTATAGGGCGTTAATGAATTAATGATTTCTCC

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siae and C. albicans (11, 17, 47, 49). However, we noted that allof these mutants, excepting the mvb12�/� strain, grew morepoorly than did the rim101�/� mutant on YPD pH 9, suggest-ing that these ESCRT pathway members have Rim101-depen-dent and -independent functions during alkaline growth. Al-though displaying an alkaline growth defect, the mvb12�/�

mutant grew slightly better than did the rim101�/� strain onthis medium, suggesting an intermediate role in Rim101-de-pendent alkaline growth. All ESCRT-I, -II, and -III mutantstested therefore displayed a growth defect on alkaline medium.Similarity in growth between these mutants and the rim101�/�mutant on YPD plus LiCl suggests that these components play

FIG. 3. FM 4-64 trafficking defects of C. albicans ESCRT mutants. (A) Exponentially growing cultures were exposed to 16 mM FM 4-64 for15 min on ice. Cells were washed with M199 pH 8, resuspended in fresh medium, and grown for 60 min at 30°C. Eighty microliters of culture wasadded to 10 �l each of 100 mM NaF and 100 mM NaN3 before microscopic examination. Strains analyzed include wild-type (WT) (DAY185),vps27�/� (DAY1160), hse1�/� (DAY1218), mvb12�/� (DAY1162), vps28�/� (DAY1161), vps36�/� (DAY1163), vps22�/� (DAY1217), vps20�/�(DAY1157), snf7�/� (DAY763), vps4�/� (DAY1155), bro1�/� (DAY1156), and doa4�/� (DAY1158) strains. Arrows denote class E-likeexclusion bodies within the cell. (B) Complementation of the vps27�/� mutant. FM 4-64 staining was performed as described above using WT(DAY185), vps27�/� (DAY1160), and vps27�/� � VPS27 (DAY1264) strains.

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only a Rim101-dependent role during LiCl growth (Fig. 4). Wenoted an exception with the mvb12�/� mutant, which grewslightly worse than did the rim101�/� mutant (Fig. 4). Thissuggests that Mvb12 contributes to LiCl growth in a Rim101-dependent and -independent manner and that this Rim101-independent function is also ESCRT independent.

We did not observe growth defects in any ESCRT-DS mu-tant strain (Fig. 4). The vps4�/�, bro1�/�, and doa4�/� strainsgrew well on YPD pH 9 and YPD plus LiCl. This is consistentwith S. cerevisiae and our results, which demonstrate thatstrains lacking VPS4 constitutively process Rim101 (17; seealso below). Further, neither the �bro1 nor the �doa4 S. cer-evisiae mutation prevents Rim101 processing, and a C. albicansbro1�/� mutant did not confer Rim101-dependent filamen-tation defects (49). These results support a model whereESCRT-DS members are not required for Rim101-dependentgrowth.

We then investigated the role of various ESCRT compo-nents in Rim101-dependent filamentation. We expected thatESCRT-I, -II, and -III would be required for Rim101 process-ing and thus have Rim101-dependent filamentation defects butthat ESCRT-0 and ESCRT-DS components would not. To test

this hypothesis, we spotted 3 �l of culture onto M199 pH 8plates and incubated them at 37°C. After 6 days, a ring offilamentation was apparent around the periphery of the wild-type colony (Fig. 5; Table 3). No peripheral filamentation wasobserved around the rim101�/� mutant. We observed wild-type filamentation around the vps27�/�, hse1�/�, bro1�/�, anddoa4�/� mutant colonies. However, we found intermediatefilamentation, indicated by a shorter filament radius or anuneven filamentous periphery, around the mvb12�/� andvps4�/� colonies, respectively. No filamentation was observedaround the vps28�/�, vps36�/�, vps22�/�, vps20�/�, orsnf7�/� mutant colonies (Fig. 5). Thus, the mutant strains withthe most severe filamentation defects were those with Rim101-dependent growth defects.

We next tested whether the ESCRT mutants affected fila-mentation at acidic pH. C. albicans does not normally filamentin acidic conditions, but we have found that both snf7�/� andvps4�/� strains had peripheral filamentation on M199 pH 4agar (47). We hypothesized that acidic filamentation may besuppressed by an ESCRT function and that other ESCRTknockout mutants would behave similarly. However, whenspotted onto M199 pH 4 plates, only the snf7�/� and vps4�/�

FIG. 4. Effect of ESCRT mutants in Rim101-dependent growth assays. Fivefold dilutions of overnight YPD cultures were spotted onto YPD,YPD pH 9, or YPD plus LiCl agar medium. Plates were incubated at 37°C for 2 days before being photographed. Strains analyzed are the sameas those in Fig. 3A.

FIG. 5. ESCRT mutant filamentation phenotypes. (Top) Three microliters of overnight YPD cultures was spotted on M199 pH 8 agarplates, and plates were incubated for 6 days at 37°C before being photographed. (Bottom) Three microliters of overnight YPD cultures wasspotted onto M199 pH 4 agar plates, and plates were incubated for 6 days at 37°C before being photographed. Strains analyzed are the sameas those in Fig. 3A.

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strains showed uniform peripheral rings after incubation at37°C (Fig. 5; Table 3). We did note smaller areas of filamen-tation in uneven patches around the periphery of the vps36�/�,vps20�/�, bro1�/�, and doa4�/� colonies. Thus, Snf7 andVps4 play the most critical roles in inhibiting acidic filamen-tation, and other ESCRT components play a small role ornone.

Finally, we tested whether the ESCRT mutations affectedserum-induced filamentation. A rim101�/� strain is unable tofilament on 4% BCS agar after 24 h of incubation at 37°C. Westreaked overnight cultures onto 4% BCS agar. After 24 h, thewild-type strain had produced a thick mesh of hyphae, whilethe rim101�/� strain was observed only in yeast form (Table3). All ESCRT-0 and ESCRT-DS mutant strains were ob-served to form wild-type-like hyphal cells, while all ESCRT-I,-II, and -III mutant strains were observed only as yeast cells(Table 3). These results support the model that ESCRT-I, -II,and -III are required for Rim101 processing in C. albicans andthat ESCRT-0 and ESCRT-DS are not.

Rim101 processing. To confirm that the growth and filamen-tation defects observed in the ESCRT mutants were or werenot associated with Rim101 processing, we introduced aRIM101-V5 allele, which encodes a functional Rim101 proteincontaining an internal V5 epitope tag, into each mutant back-ground. Strains were grown to mid-log phase, and protein wascollected for Western blot analysis. After growth in M199 pH8 medium, we observed distinct Rim101-V5 bands in the wild-type strain: the full-length, unprocessed 84-kDa band (FL), theprocessed, active 74- to 78-kDa bands (P1), and the processed65-kDa band of unknown function (P2) (Fig. 6A) as reportedpreviously (25). We noted similar banding patterns in the twoESCRT-0 knockout strains, the vps27�/� and hse1�/� strains(Fig. 6A). All ESCRT-I, -II, and -III knockout strains showedlittle to no processing to the P1 or P2 forms, indicating thatRim101 processing is defective in these strains (Fig. 6A).The vps4�/�, bro1�/�, and doa4�/� strains, whose productsall act downstream of Snf7 recruitment to the endosome,showed a banding pattern similar to that of the wild-type

strain (Fig. 6A). Since the �vps4 mutant in S. cerevisiaeprocesses Rim101 under noninducing conditions (17), weanalyzed the RIM101-V5 processing in the ESCRT mutantsafter growth in M199 pH 4 medium. In wild-type cells, onlyfull-length Rim101 was observed. Similar results were observed inall mutant strains except the vps4�/� mutant strain (Fig. 6B). Thevps4�/� strain displayed both P1 and P2 bands at pH 4, indicating

TABLE 3. Phenotypic analyses of ESCRT mutantsa

Strain Genotype

Phenotypeb

% WT FaDu celldamage

Growth onmedium type: Filamentation on:

BPSgrowth

Rim101processing

Alkaline LiCl Alkalineagar

Acidicagar BCS agar

DAY185 WT � � � � � � � NADAY25 rim101�/� � � � � � � � 3 � 5DAY1160 vps27�/� � � � � � � � 63 � 17DAY1218 hse1�/� � � � � � � � 94 � 16DAY1162 mvb12�/� � � � � � � � 37 � 15DAY1161 vps28�/� � � � � � � � 12 � 7DAY1163 vps36�/� � � � � � � � 6 � 6DAY1217 vps22�/� � � � � � � � 8 � 8DAY1157 vps20�/� � � � � � � � 14 � 3DAY763 snf7�/� � � � � � � � 4 � 3DAY1155 vps4�/� � � � � � � � 59 � 7DAY1156 bro1�/� � � � � � � � 68 � 12DAY1158 doa4�/� � � � � � � � 68 � 11

a WT, wild type; NA, not applicable.b �, positive; �, negative; �, intermediate.

FIG. 6. Rim101 processing at alkaline and acidic pH. (A) Over-night YPD cultures were inoculated into 40 ml M199 pH 4 andgrown for 5 h at 30°C. Cell pellets were collected and resuspendedin 50 ml M199 pH 8 and grown for 30 min at 30°C, protein purified,and analyzed by Western blotting. Rim101-V5 was detected after 1 h ofincubation with anti-V5-HRP antibody (Invitrogen) by chemilumines-cence. Strains analyzed included wild-type (WT) (DAY1212), vps27�/�(DAY1178), hse1�/� (DAY1221), mvb12�/� (DAY1193), vps28�/�(DAY1183), vps36�/� (DAY1188), vps22�/� (DAY1222), vps20�/�(DAY1257), snf7�/� (DAY568), vps4�/� (DAY576), bro1�/�(DAY1219), and doa4�/� (DAY1260) strains. Molecular mass markers(in kilodaltons) are indicated to the right. (B) Overnight YPD culture wasinoculated into 40 ml M199 pH 4 and grown for 5 h at 30°C. Proteinpreparations and Western blot analysis were done as described above.

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improper Rim101 activation. However, the other ESCRT-DSmutation did not affect Rim101 processing at pH 4. Regardless,the growth defects observed in the ESCRT mutants correlatedstrongly with the ability of the mutant strains to process Rim101at pH 8.

Iron-dependent growth defects. Rim101 promotes expres-sion of genes involved in uptake of iron, which is insoluble inalkaline environments (5). Studies of several C. albicans ES-CRT mutants demonstrated that members of ESCRT-I, -II,and -III are required for transporting hemoglobin to the vac-uole, including the ESCRT-III member VPS2 (44). SinceRim101 is not required for vacuolar transport, we hypothe-sized that the ESCRT pathway may play a role in iron acqui-sition independent of Rim101 processing.

To test this hypothesis, we grew strains in YPD plus 50 �Miron chelator BPS and serially diluted them onto YPD or YPDplus 150 �M BPS agar medium. After 3 days of growth, allstrains grew equally well on YPD medium. The wild-type straingrew well on YPD plus BPS plates, resulting in large thickcolonies. The ftr1�/� mutant strain, which lacks a high-affinityiron permease (35), did not grow on YPD plus BPS medium,while the rim101�/� mutant strain showed an intermediategrowth defect, able to form only thin colonies on the medium.Similar intermediate growth defects were seen in the vps27�/�,hse1�/�, snf7�/�, vps4�/�, bro1�/�, and doa4�/� mutants(Fig. 7), although a more severe growth defect was observedwith the vps28�/�, vps36�/�, vps22�/�, and vps20�/� mutantstrains (Fig. 7). The mvb12�/� mutant strain grew similarly tothe wild-type strain, which likely reflects the limited pheno-types observed with this mutant (Fig. 3, 4, and 5). SinceESCRT-I, ESCRT-II, and ESCRT-III mutants have more severegrowth defects than do ESCRT-0 and ESCRT-DS mutants,this suggests that the ESCRT pathway contributes to iron ac-quisition through Rim101-dependent and -independent mech-anisms. Furthermore, since the snf7�/� mutant showed a

growth defect similar to that of the rim101�/�, ESCRT-0, andESCRT-DS mutants, this suggests that the snf7�/� mutationdoes not affect both Rim101-dependent and ESCRT-depen-dent iron acquisition.

Epithelial cell damage. Epithelial cells line the skin and allmucosal surfaces and are the primary cell type on which C.albicans resides as a commensal, and Rim101 activation isrequired to damage epithelial cells (29). C. albicans causesdamage to the epithelia by mediating its own endocytosis bythe epithelial cells following germination and causing epithelialcell lysis (31, 34). With radiolabeling of a monolayer of FaDuoropharyngeal epithelial cell culture, damage can be moni-tored by measuring the radioactivity released to the culturesupernatant. We used our ESCRT mutants to ask the question:does the ESCRT pathway play a Rim101-independent role indamaging epithelial cells?

When incubated with wild-type C. albicans, we observedrobust epithelial cell damage, with a typical assay resulting in30 to 50% specific Cr51 release (14). To facilitate comparisonbetween assays, we normalized the wild-type damage level ineach assay and compared the amount of damage induced bythe mutant strains with that induced by the wild type. Asreported previously, the rim101�/� mutant showed a severereduction in epithelial damage (Fig. 8A), yielding �3% ofwild-type damage. While we found that the ESCRT-0 hse1�/�mutant was not statistically different from the wild type, thevps27�/� mutant had an �35% decrease in wild-type damage(P 0.001). We observed significant decreases in epithelialcell damage with all ESCRT-I, -II, and -III mutants, althoughthe mvb12�/� mutant caused more damage than did the otherESCRT-I member. The vps4�/�, bro1�/�, and doa4�/� strainsall showed an �35 to 40% decrease in damage compared tothe wild type, like the vps27�/� strain. The vps27�/�, bro1�/�,and doa4�/� strains did not affect Rim101-dependent pheno-types or Rim101 processing but did affect FM 4-64 trafficking,demonstrating that ESCRT function plays a role in C. albicans-mediated epithelial cell damage independent of Rim101 acti-vation.

OPC mouse model of disease. We next determined the con-tribution of ESCRT function to virulence using an oropharyn-geal candidiasis (OPC) mouse model. Cortisone-immunosup-pressed mice were infected orally with wild-type, rim101�/�,vps27�/�, or snf7�/� C. albicans. Ten mice per strain weresacrificed at 3 days postinfection (DPI) and at 6 DPI. Mousetongues were harvested to assess oral fungal burden, and micewere weighed daily. This approach allowed us to assess fungalburden and mouse morbidity.

We first assessed the fungal burden of mice infected with5 � 103 CFU. At 3 DPI, the oral cavity of mice infected withthe wild-type strain showed an average fungal burden of 4 �104 CFU/g tissue. A significant decrease in fungal burden wasobserved between the wild-type strain and the three C. albicansmutants (P 0.04 for rim101�/�, vps27�/�, and snf7�/�strains) (Fig. 9A). Mice infected with the vps27�/� strain car-ried 1 log less CFU/g tongue compared to mice infected withthe wild type; the rim101�/� and snf7�/� strains had nearly a2-log-lower burden than did wild-type C. albicans. The fact thatall mutants colonized to lower burden levels suggests that allthree genes play a role in C. albicans pathogenesis. Addition-ally, both the rim101�/� and snf7�/� strains colonized the oral

FIG. 7. ESCRT mutants have growth defects on iron-depleted me-dium. YPD plus BPS cultures were diluted 1:50 in PBS, and 5-fold serialdilutions were spotted onto YPD and YPD plus BPS agar plates. Plateswere incubated at 37°C for 2 days before being photographed. Strainsinvestigated included wild-type (WT) (DAY185), ftr1�/� (DAY750),rim101�/� (DAY25), vps27�/� (DAY1160), hse1�/� (DAY1218),mvb12�/� (DAY1162), vps28�/� (DAY1161), vps36�/� (DAY1163),vps22�/� (DAY1217), vps20�/� (DAY1157), snf7�/� (DAY763),vps4�/� (DAY1155), bro1�/� (DAY1156), and doa4�/� (DAY1158)strains.

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cavity at a significantly lower burden than did the vps27�/�strain (P 0.04 and 0.03, respectively) (Fig. 9A). Thus, bothRim101 processing and ESCRT function are necessary forwild-type levels of in vivo tongue colonization, but defects inRim101 processing result in more severe virulence defects thando defects in ESCRT function.

At 6 DPI, all mice infected with the wild-type strain were stillcolonized, although the fungal burden decreased by approxi-mately a log (4 � 103 CFU/g tissue). All mice infected with therim101�/� and snf7�/� mutants and all but two mice infectedwith the vps27�/� mutant cleared the infection to levels belowdetection (Fig. 9A). This indicates that both Rim101 process-

FIG. 8. (A) ESCRT mutants show defects in epithelial cell damage. C. albicans cells (105) were incubated for 10 h with 51Cr-labeled FaDu cells.Supernatant was collected and compared to that of uninfected FaDu cells. Samples were run in triplicate for each assay. Graph shows damageconferred by each mutant relative to a simultaneously tested wild-type (WT) strain. Mutants were tested in at least three separate assays. *, P 0.05. Strains analyzed included wild-type (DAY185), ftr1�/� (DAY750), rim101�/� (DAY25), vps27�/� (DAY1160), hse1�/� (DAY1218),mvb12�/� (DAY1162), vps28�/� (DAY1161), vps36�/� (DAY1163), vps22�/� (DAY1217), vps20�/� (DAY1157), snf7�/� (DAY763), vps4�/�(DAY1155), bro1�/� (DAY1156), and doa4�/� (DAY1158) strains. (B) Complementation of the vps27�/� mutant using wild-type (DAY185),vps27�/� (DAY1160), and vps27�/� � VPS27 (DAY1264) strains.

FIG. 9. A vps27�/� mutant is attenuated in a mouse model of oral candidiasis. (A) Tongue fungal burden and animal weight loss after low-doseinoculation. Cortisone-immunosuppressed animals were infected with 50 �l of 105 CFU/ml of wild-type (WT) (DAY185), rim101�/� (DAY25),vps27�/� (DAY1160), and snf7�/� (DAY763) C. albicans. At 3 DPI half the animals were harvested and their tongues were collected fordetermination of fungal burden. The remaining animals were reimmunosuppressed with cortisone and harvested at 6 DPI for determination offungal burden. *, P 0.05. (B) Tongue fungal burden and animal weight loss after high-dose inoculation. The experiment was run identically butwith an infectious dose of 50 �l of 106 CFU/ml C. albicans.

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ing and ESCRT function are necessary for maintaining infec-tion and that Rim101 processing plays a more important role inmaintenance than ESCRT function.

During the course of infection, we weighed the mice as ameasure of mouse morbidity. Mice with oral candidiasis canlose weight quickly, and mice infected with the four C. albicansstrains lost weight over the course of the experiment. However,by 6 DPI, mice infected with the wild-type strain had lostsignificantly more weight than did mice infected with any of thethree mutant strains (P 0.003, P 0.03, and P 0.01 forrim101�/�, vps27�/�, and snf7�/� strains, respectively) (Fig.9A). There was no statistical difference in weight loss betweenmice infected with the rim101�/�, vps27�/�, and snf7�/� strains.This suggests that weight loss correlates with fungal burden andthat mice more heavily colonized will manifest disease symptomsmore quickly than those less heavily colonized.

Since the mutant strains showed a decreased burden at 3DPI, we hypothesized that this might be due to poor initialcolonization, which might be overcome with a higher initialinoculum. To address this possibility, we infected mice with a10-fold-higher inoculum (5 � 104 CFU) and assessed fungalburden at 3 DPI and 6 DPI. At 3 DPI, the oral cavity of miceinfected with the wild-type strain showed an average fungalburden of 6 � 104 CFU/g tissue, which was comparable to thecolonization level for the lower dose (Fig. 9B). Mice infectedwith the rim101�/� strain showed a significant decrease inburden, 2 � 104 CFU/g tissue, compared to those infected withwild-type cells (P 0.03). However, the rim101�/� strain-infected mice at the higher dose were colonized with a 2-log-higher burden than were the rim101�/� strain-infected mice atthe lower dose. Mice infected with the vps27�/� strain alsoshowed �2 logs more colonization than did mice infected withthe lower dose of the vps27�/� strain. Unlike the rim101�/�mutant, the vps27�/� mutant had an �2-fold-higher averagefungal burden, 2 � 105 CFU/g tissue, than did mice infectedwith wild-type C. albicans (Fig. 9B). Mice infected with thesnf7�/� strain showed a significant decrease in fungal burdenfrom that of wild-type-infected mice (P 0.005), compara-ble to that of the snf7�/� strain-infected mice in the lower-dose experiment. Because an increase in the inoculum ofwild-type C. albicans did not increase the fungal burden at 3DPI, we deduce that there are a finite number of sites withinthe mouse oral cavity for C. albicans to reside in.

At 6 DPI, mice infected with the wild-type strain exhibited afungal burden of 1 � 105 CFU/g tissue. Mice infected with therim101�/� or vps27�/� strain showed no significant differencefrom wild-type-infected mice in oral fungal burden with anaverage of 8 � 104 and 9 � 104 CFU/g, respectively (P 0.50and P 0.15, respectively) (Fig. 9B). This suggests thatRim101 processing and ESCRT function contribute indepen-dently to maintaining infection. All mice but one cleared thesnf7�/� mutant from their oral cavity by 6 DPI, indicating thatthe effects of Rim101 processing and ESCRT function areadditive. Alternatively, the snf7�/� mutant may have addi-tional defects independent of either Rim101 or ESCRT func-tion that decrease the viability of this strain. Since increasingthe inoculum of the rim101�/� and vps27�/� mutants, but notthe snf7�/� mutant, improved establishment and maintenanceof infection, we conclude that the rim101�/� and vps27�/�strains have defects in initial oral colonization.

We also monitored weight loss during the course of theinfection with the higher inocula. Again, mice lost weight overthe course of the infection, except for snf7�/� strain-infectedmice (Fig. 9B). While rim101�/� strain- and vps27�/� strain-infected mice lost weight, mice infected with wild-type C. al-bicans lost a significantly larger amount of weight than didmice infected with either mutant (P 0.001, P 0.0005, andP 0.0005 for rim101�/�, vps27�/�, and snf7�/� mutants,respectively) (Fig. 9B). Although mice infected with the wild-type, rim101�/�, or vps27�/� strains are colonized to similarlevels (Fig. 9B), mice infected with wild-type C. albicans showsignificantly more morbidity than do mice infected with eithermutant. These results demonstrate that the ESCRT pathwayplays a Rim101-independent role during C. albicans pathogen-esis.

VPS27 complementation. To confirm that the vps27�/� phe-notypes observed were due to VPS27 specific mutation, wetransformed the vps27�/� strain with a VPS27-containingcomplementation plasmid and compared it to the prototrophicvps27�/� mutant. Addition of VPS27 to the vps27�/� mutantcompletely restored the FM 4-64 trafficking phenotype to awild-type staining pattern (Fig. 3B) and restored epithelial celldamage defect compared to the vps27�/� strain (P 0.01)(Fig. 8B). However, the complemented strain did not restoreepithelial cell damage to wild-type levels, nor did it restoregrowth on iron-limiting medium to wild-type levels (Fig. 7),suggesting that complementation is not complete. This partialrescue could be due to position effects, haploinsufficiency, orinsufficient promoter sequence inclusion in the construct.However, the complementation of the epithelial cell damageand FM 4-64 trafficking phenotypes led us to conclude that thephenotypes associated with the vps27�/� mutant are indeeddue to loss of VPS27.

DISCUSSION

We have used a series of ESCRT pathway mutants to inves-tigate the role of individual ESCRT protein components inboth Rim101 processing and MVB trafficking in C. albicans.We found that ESCRT-I, -II, and -III components, but notESCRT-0 and ESCRT-DS components, are required for pro-teolytic processing of the transcription factor Rim101. Our invitro epithelial cell damage studies demonstrate that ESCRTfunction plays a Rim101-independent role in epithelial celldamage, and our in vivo studies suggest that ESCRT functionis required for wild-type C. albicans pathogenesis. We foundthat Vps27 is required for wild-type colonization levels whenorally administered to mice at a relatively low dose but that thiscolonization defect can be overcome by administering a higherdose. However, even when mice are colonized with similarnumbers of wild-type and vps27�/� C. albicans, wild-type-in-fected mice lose significantly more weight than do knockout-infected mice. Thus, ESCRT function is required for normalcolonization and morbidity associated with C. albicans infec-tion independent of Rim101.

Since the phenotypes of our ESCRT pathway mutants wereinternally consistent in general (see below) and based on thefact that our ESCRT mutants act similarly to ESCRT mutantsin other systems, we conclude that the genes that we identifiedencode bona fide ESCRT pathway members. This idea is sup-

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ported by the complementation test of the vps27�/� mutant,which restored the FM 4-64 trafficking and epithelial cell dam-age phenotypes. However, we did note that growth phenotypesdiffered between mutants in the ESCRT-I complex membersVps28 and Mvb12. While the vps28�/� mutant behaved simi-larly to the ESCRT-II mutants, the mvb12�/� mutant hadintermediate phenotypes for alkaline growth and filamentationand more severe defects on LiCl-supplemented medium. Thissuggests that Mvb12 may play an accessory role in the ESCRTpathway and potentially have ESCRT-independent pheno-types, although further analyses are necessary to understandMvb12 function. We also noted differences between mutants inthe ESCRT-0 members Vps27 and Hse1. While the vps27�/�mutant conferred defects in epithelial cell damage, thehse1�/� mutant did not (Fig. 8). To the best of our knowledge,this is the first demonstration that Hse1 is not necessary for allVps27-dependent phenotypes. Numerous analyses have beenperformed using a mutation within a single ESCRT complexsubunit as representative of the behavior of the complex. Thephenotypic differences between the mvb12�/� and vps28�/�mutant strains and vps27�/� and hse1�/� mutant strains stressthe hazard in this assumption. Our data highlight the fact thatmolecular subunits of a complex do not necessarily have thesame effect on function, and valuable information may bemissed by using a single representative mutation.

Are all ESCRT complexes required for Rim101 processing?ESCRT complex function plays a role in activating the tran-scription factor Rim101 by recruiting the processing machineryto the endosomal membrane in a number of fungi, including C.albicans, S. cerevisiae, and Aspergillus fumigatus (10, 37). In S.cerevisiae, the ESCRT-0 member Vps27 does not play a rolein Rim101 activation (49), suggesting that the same mayhold true in C. albicans. Our studies clearly establish thatneither Vps27 nor Hse1, the other ESCRT-0 member, isrequired for Rim101-dependent growth or Rim101 processing(Fig. 2 and 4). While it is possible that C. albicans containsredundant VPS27 or HSE1 homologs, this is unlikely, as bothvps27�/� and hse1�/� strains displayed FM 4-64 traffickingpatterns associated with ESCRT defects. Additionally, neitherVPS27 nor HSE1 paralogs were identified through BLASTsearch analyses. Thus, ESCRT-0 is not required for Rim101processing in C. albicans.

Why does Rim101 processing bypass ESCRT-0 yet requireESCRT-I, -II, and -III? One possibility is that ESCRT-I, -II,and -III are recruited directly by the upstream Rim101 sig-naling molecules, Rim21 and Rim8. Rim21/PalH, a trans-membrane protein found at the plasma membrane, interactswith ubiquitinated Rim8/PalF in Aspergillus nidulans (19),and Rim8 ubiquitination has recently been documented in S.cerevisiae (18). Further, ubiquitinated Rim8 can interact di-rectly with ESCRT-I member Vps23 (18). The Rim21-Rim8complex is thought to act as a surrogate “receptor” in alkalineconditions, initiating endocytosis and subsequent ESCRT com-plex assembly. This suggests that the Rim21-Rim8 complexbypasses ESCRT-0 by recruiting ESCRT-I directly. ESCRT-Irecruitment appears to be mediated through Rim8 mimicry ofESCRT-0 component Vps27, as S. cerevisiae Rim8 interactswith Vps23 through an SXP motif similar to the Vps27 PSAPmotif which interacts with Vps23 (18). However, Rim8 ubiq-uitination in S. cerevisiae is pH independent and thus does

not explain how the cell can activate Rim101 only underneutral-alkaline conditions. Other systems are known to re-cruit ESCRT-I directly, bypassing ESCRT-0, to assemblethe core ESCRT complexes at diverse cellular locations fordistinct functions such as cytokinesis and viral budding (6,26, 27). Direct recruitment of ESCRT-I by Rim101 signalingmembers is likely conserved across fungi containing Rim101.

ESCRT function in pathogenesis. Previous studies from ourlab analyzed the effects of a variety of snf7 alleles on C. albi-cans-stimulated epithelial cell damage (47). These studies sug-gested that the only contribution of Snf7 to epithelial celldamage is Rim101 dependent. However, these alleles are orare likely to be partially functional; thus, the damage assay maynot be sensitive enough to detect differences with these partialdisruptions. Thus, our previous experiments included severalcaveats and were not wholly conclusive.

The vps27�/�, hse1�/�, vps4�/�, bro1�/�, and doa4�/� mu-tant strains were all able to grow like the wild type on Rim101-dependent media, and all but the vps4�/� strain presentednormal Rim101-dependent filamentation. Thus, we expectedthese strains to be the most representative of independentESCRT function during C. albicans pathogenesis. The similardecreases in the vps27�/�, bro1�/�, and doa4�/� mutants inthe FaDu epithelial cell damage assay suggest that the defect maybe due to the common ESCRT function of these genes. Theslightly larger decrease of the vps4�/� mutant may be due to thedysregulation of Rim101 processing in this strain (Fig. 6).

Previous studies have looked at the role of ESCRT complexproteins in C. albicans infection by using a bloodstream infec-tion mouse model (12). The ESCRT complex members previ-ously studied, Vps28 and Snf7/Vps32, function in both Rim101processing and MVB trafficking. These mutant strains werefound to be less pathogenic than the rim101�/� strain.These findings suggested that ESCRT function plays a rolein C. albicans pathogenesis, but that study did not truly studyESCRT function independently from Rim101 function. Thevps27�/� and hse1�/� strains used here study ESCRT func-tion wholly uncoupled from Rim101 processing.

Data from the OPC mouse model demonstrate that theESCRT pathway plays a Rim101-independent role in patho-genesis. At a lower inoculum, the vps27�/� strain was defectivein colonization. This defect was overcome with an increasedinoculum, but similar fungal burdens were not an indication ofsimilar disease states, as mice infected with wild-type C. albi-cans lost significantly more weight than did mice infected withthe vps27�/� strain. Thus, ESCRT plays a role both in estab-lishing colonization during infection (at low doses) and indisease-associated morbidity (at high doses).

We compared the wild-type and vps27�/� strains in sev-eral assays for filamentation, adherence, and growth rateand observed no differences to account for morbidity differ-ences (data not shown). Previous studies have shown thatmutation of ESCRT complexes can increase sensitivity to thecell wall-targeting drug caspofungin more than can mutation ofRIM101 alone (12), suggesting that the ESCRT pathway playsa role in cell wall function. The fungal cell wall initiates contactwith host cells, resulting in damage (34), and may explain thedamage defects of the ESCRT mutants. Additionally, we andothers have shown that the ESCRT pathway is required foracquiring nutrients, such as iron (44). The role played by the

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ESCRT pathway during infection is therefore likely to be mul-tifactorial, affecting both cell wall architecture and nutrientacquisition.

C. albicans niche size. Are fungal colonization levels depen-dent on inoculum concentration? Lower- and higher-dose in-ocula lead to similar fungal burdens with the wild-type strain,while a higher-dose inoculum leads to a higher fungal burdenwith the mutant strains. However, mutant strain burden didnot increase much beyond wild-type levels, if at all (Fig. 9).This potential upper limit of fungal colonization suggests thatthere exist a finite number of sites where C. albicans can growwithin its host.

We were able to rescue colonization defects of both thevps27�/� and rim101�/� strains to wild-type levels by increas-ing the inoculum dose. However, we observed similar wild-typecolonization numbers at 3 DPI in animals infected with low- orhigh-dose inocula. These facts indicate that C. albicans has alimited niche size in the oral cavity. We manipulated the nichesize of our animals with cortisone and tetracycline treatments,and different manipulations could further alter this niche size.However, given a set of conditions in its host, there is an upperthreshold to the C. albicans burden that it can bear.

Our studies have shown that C. albicans requires a fullyfunctional ESCRT complex for wild-type pathogenic levelsduring in vitro and in vivo pathogenesis. Understanding theESCRT complex function in C. albicans presents opportunitiesto molecularly characterize a nontraditional signal transduc-tion cascade and to investigate the mechanism of ESCRTfunction during C. albicans interactions with the host.

ACKNOWLEDGMENTS

We thank the members of the Davis lab and Kirsten Nielson’s lab forhelpful discussions on this work. We thank Laura Okagaki for micros-copy help.

The project described was supported by the NIH National Instituteof Allergy and Infectious Disease award R01-AI064054-01, by NIHT32DE007288 from the National Institute of Dental and CraniofacialResearch, and by the Investigators in Pathogenesis of Infectious Dis-ease Award from the Burroughs Wellcome Fund.

The content is solely the responsibility of the authors and does notnecessarily represent the official views of the National Institute ofDental and Craniofacial Research or the National Institutes of Health.

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Structure and membrane remodeling activity of ESCRT-III ... · ATPases. ESCRT-III assemblies promote membrane constriction and fission, possibly in concert with VPS4. Humans express

Structure and membrane remodeling activity of ESCRT-III ... · ATPases. ESCRT-III assemblies promote membrane constriction and fission, possibly in concert with VPS4. Humans express

The ESCRT machinery: From the plasma membrane …audhyalab.org/admin/resources/schuh-and-audhya-2014.pdfunderstanding about how they associate with one another. Combined with genetic-

The ESCRT machinery: From the plasma membrane …audhyalab.org/admin/resources/schuh-and-audhya-2014.pdfunderstanding about how they associate with one another. Combined with genetic-

Author Manuscript NIH Public Access 1,3, Eiji Morita 1 ...cheetah.biochem.utah.edu/uploads/7/9/8/6/7986331/kieffer2008devcell.pdfTwo distinct modes of ESCRT-III recognition are required

Author Manuscript NIH Public Access 1,3, Eiji Morita 1 ...cheetah.biochem.utah.edu/uploads/7/9/8/6/7986331/kieffer2008devcell.pdfTwo distinct modes of ESCRT-III recognition are required

RESEARCH ARTICLE Open Access Identification of conserved ...€¦ · have been identified thus far: (i) the PT/SAP motif re-cruits the ESCRT-1 component Tsg101 (Tumor Suscep-tibility

RESEARCH ARTICLE Open Access Identification of conserved ...€¦ · have been identified thus far: (i) the PT/SAP motif re-cruits the ESCRT-1 component Tsg101 (Tumor Suscep-tibility

Genome-wide RNAi screen reveals a role for the …genepath.med.harvard.edu/perrimon/papers/2013_PNAS_Silva-Ayala... · Genome-wide RNAi screen reveals a role for the ESCRT complex

Genome-wide RNAi screen reveals a role for the …genepath.med.harvard.edu/perrimon/papers/2013_PNAS_Silva-Ayala... · Genome-wide RNAi screen reveals a role for the ESCRT complex

Ferroptotic pores induce Ca2+ fluxes and ESCRT-III …Jimenez & Perez, 2017). The Endosomal sorting complexes required for transport (ESCRT) machinery seems to play a critical counterbalancing

Ferroptotic pores induce Ca2+ fluxes and ESCRT-III …Jimenez & Perez, 2017). The Endosomal sorting complexes required for transport (ESCRT) machinery seems to play a critical counterbalancing

Crystallographic and Functional Analysis of the ESCRT-I /HIV-1 …myweb.chonnam.ac.kr/~stbiochm/Publications/PDF/[2010... · 2014-12-15 · Structure Article Crystallographic and

Crystallographic and Functional Analysis of the ESCRT-I /HIV-1 …myweb.chonnam.ac.kr/~stbiochm/Publications/PDF/[2010... · 2014-12-15 · Structure Article Crystallographic and

ESCRT-II controls retinal axon growth by regulating DCC ...rsob.royalsocietypublishing.org/content/royopenbio/6/4/150218.full.pdf · cytosis and local protein synthesis (LPS) [5]

ESCRT-II controls retinal axon growth by regulating DCC ...rsob.royalsocietypublishing.org/content/royopenbio/6/4/150218.full.pdf · cytosis and local protein synthesis (LPS) [5]

The Interplay between ESCRT and Viral Factors in the ...

The Interplay between ESCRT and Viral Factors in the ...

Candida albicans RIM101 pH Response Pathway Is Required for … · The RIM101 pathway controls gene expression and the yeast-to-hyphal transition in C. albicans in response to changes

Candida albicans RIM101 pH Response Pathway Is Required for … · The RIM101 pathway controls gene expression and the yeast-to-hyphal transition in C. albicans in response to changes

Cryptococcus neoformans Rim101 Is Associated with Cell Wall … · Cryptococcus neoformans Rim101 Is Associated with Cell Wall Remodeling and Evasion of the Host Immune Responses

Cryptococcus neoformans Rim101 Is Associated with Cell Wall … · Cryptococcus neoformans Rim101 Is Associated with Cell Wall Remodeling and Evasion of the Host Immune Responses

Role of Mitochondrial Retrograde Pathway in Regulating Ethanol … · 2017. 4. 13. · RAS-cAMP, Snf1, Rpd3(L), and Rim101] but required the mitochondrial retrograde (RTG) pathway,

Role of Mitochondrial Retrograde Pathway in Regulating Ethanol … · 2017. 4. 13. · RAS-cAMP, Snf1, Rpd3(L), and Rim101] but required the mitochondrial retrograde (RTG) pathway,

Structure of the ESCRT-II endosomal trafficking complex

Structure of the ESCRT-II endosomal trafficking complex

Biochimica et Biophysica Acta - Kanazawa Universitybiophys.w3.kanazawa-u.ac.jp/References/ATPases/membrane...pathway. Flat clathrin together with GGA proteins and ESCRT-0 (Vps27-Hse1)

Biochimica et Biophysica Acta - Kanazawa Universitybiophys.w3.kanazawa-u.ac.jp/References/ATPases/membrane...pathway. Flat clathrin together with GGA proteins and ESCRT-0 (Vps27-Hse1)

Supplemental Information Recruitment of UBPY and ESCRT ...Supplemental Movie 2, related to Figure 1(available online) Supplemental Movie 3, related to Figure 1(available online) Supplemental

Supplemental Information Recruitment of UBPY and ESCRT ...Supplemental Movie 2, related to Figure 1(available online) Supplemental Movie 3, related to Figure 1(available online) Supplemental

Emily Zhen Guo1 and Zhaohui Xu1,2 - Journal of Biological … · 2015. 2. 5. · Distinct Mechanisms of Recognizing Endosomal Sorting Complex Required for Transport (ESCRT)-III Protein

Emily Zhen Guo1 and Zhaohui Xu1,2 - Journal of Biological … · 2015. 2. 5. · Distinct Mechanisms of Recognizing Endosomal Sorting Complex Required for Transport (ESCRT)-III Protein

Dual roles of an Arabidopsis ESCRT component FREE1 ...Dual roles of an Arabidopsis ESCRT component FREE1 in regulating vacuolar protein transport and autophagic degradation Caiji Gao

Dual roles of an Arabidopsis ESCRT component FREE1 ...Dual roles of an Arabidopsis ESCRT component FREE1 in regulating vacuolar protein transport and autophagic degradation Caiji Gao

CELL BIOLOGY ATP-dependent force generation …...CELL BIOLOGY ATP-dependent force generation and membrane scission by ESCRT-III and Vps4 Johannes Schöneberg1,2,3, Mark Remec Pavlin2,4*,

CELL BIOLOGY ATP-dependent force generation …...CELL BIOLOGY ATP-dependent force generation and membrane scission by ESCRT-III and Vps4 Johannes Schöneberg1,2,3, Mark Remec Pavlin2,4*,

Cryptococcus neoformans Requires the ESCRT Protein Vps23 for … · mune system to maintain low iron availability. ... bacteria(22,23,24).Gram-negativebacteria,forexample,gener-ally

Cryptococcus neoformans Requires the ESCRT Protein Vps23 for … · mune system to maintain low iron availability. ... bacteria(22,23,24).Gram-negativebacteria,forexample,gener-ally

G Model ARTICLE IN PRESSPlease cite this article in press as: E.B. Frankel, A. Audhya, ESCRT-dependent cargo sorting at multivesicular endosomes, Semin Cell Dev Biol (2017), ...

G Model ARTICLE IN PRESSPlease cite this article in press as: E.B. Frankel, A. Audhya, ESCRT-dependent cargo sorting at multivesicular endosomes, Semin Cell Dev Biol (2017), ...