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A tethering coherent protein in autophagosome maturationDandan Chen and Qing Zhong*Division of Biochemistry, Biophysics and Structural Biology; Department of Molecular and Cell Biology; University of California at Berkeley; Berkeley, CA USA

Keywords: autophagy, TECPR1,ATG12ATG5, autophagosomematuration

Submitted: 04/02/12

Revised: 04/02/12

Accepted: 04/03/12http://dx.doi.org/10.4161/auto.20255

*Correspondence to: Qing Zhong;

Email: qingzhong@berkeley.edu

Punctum to: Chen D, Fan W, Lu Y, Ding X, Chen S,

Zhong Q. A mammalian autophagosome

maturation mechanism mediated by TECPR1 and

the Atg12-Atg5 conjugate. Mol Cell 2012;

45:62941; PMID:22342342; http://dx.doi.org/10.

1016/j.molcel.2011.12.036

Autophagy is a cellular pathway thatdegrades damaged organelles, cyto-sol and microorganisms, thereby main-taining human health by preventing

various diseases including cancers, neuro-degenerative disorders and diabetes. Inautophagy, autophagosomes carryingcellular cargoes fuse with lysosomes fordegradation. The proper autophagosome-lysosome fusion is pivotal for efficientautophagy activity. However, the mole-cular mechanism that specifically directsthe fusion process is not clear. Ourstudy reported that lysosome-localizedTECPR1 (TECtonin -Propeller Repeatcontaining 1) binds the autophagosome-localized ATG12ATG5 conjugate andrecruits it to autolysosomes. TECPR1also binds PtdIns3P in an ATG12

ATG5-dependent manner. Conse-quently, depletion of TECPR1 leadsto a severe defect in autophagosomematuration. We propose that the inter-action between TECPR1 and ATG12

ATG5 initiates the fusion betweenthe autophagosome and lysosome, andTECPR1 is a TEthering CoherentPRotein in autophagosome maturation.

ATG5, a 32 kDa protein essential forautophagy, is covalently modified by

ATG12. The ATG12

ATG5 conjugateinteracts non-covalently with ATG16 toform the ATG12ATG5-ATG16 complexthat localizes to autophagosome precursorsand plays an essential role in autophago-some formation. Besides ATG16, another

ATG12ATG5T-associated protein,TECPR1, has been discovered recently.TECPR1 was first described as a com-ponent of the autophagy interactionnetwork and reported to associate with

the ATG12ATG5-ATG16 complex,CCT chaperonin complex and TRAPPvesicle-tethering complex. However, ourimmunoprecipitation (IP) result showedthat TECPR1 could pull down ATG12

ATG5 but not ATG16, suggesting thatTECPR1 and ATG16 form two mutuallyexclusive protein complexes with ATG12

ATG5. With deletion mutagenesis andco-IPs, we identified a region of TECPR1spanning from amino acids 566 to 610 asthe ATG12ATG5 interacting region(AIR), which is necessary and sufficientto bind to ATG12ATG5.

To investigate the subcellular localiza-tion of TECPR1, we set up an induciblecell line in which tagged TECPR1 isexpressed at a physiological level controlledby doxycycline addition. We found that

tagged TECPR1 mainly localizes to lyso-somes but not early autophagic mem-branes. And once autolysosomes areaccumulated by treatment with chloro-quine (CQ), a lysosomal inhibitor, thecolocalization between TECPR1 and LC3is significantly increased. Using immuno-EM, we observed that EGFP-TECPR1signals are present on the membrane of thelarge autolysosomes in CQ-treated cells.These results indicate that TECPR1resides on the autolysosome membrane.Most importantly, we found that ATG5 is

recruited to the autolysosome membraneby TECPR1, which extends the currentautophagy model that ATG5 localizesexclusively to phagophores.

We also investigated the function ofTECPR1 in autophagy by RNAi knock-down experiments. The autophagy pheno-types we observed in TECPR1-deficientcells suggest a role of TECPR1 inautophagosome maturation. In TECPR1-deficient cells, two well-known autophagic

AUTOPHAGIC PUNCTUM

Autophagy 8:6, 985986; June 2012; G 2012 Landes Bioscience

www.landesbioscience.com Autophagy 985

http://dx.doi.org/10.4161/auto.20255http://www.ncbi.nlm.nih.gov/pubmed/22342342http://dx.doi.org/10.1016/j.molcel.2011.12.036http://dx.doi.org/10.1016/j.molcel.2011.12.036http://dx.doi.org/10.1016/j.molcel.2011.12.036http://dx.doi.org/10.1016/j.molcel.2011.12.036http://www.ncbi.nlm.nih.gov/pubmed/22342342http://dx.doi.org/10.4161/auto.20255http://dx.doi.org/10.4161/auto.20255

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substrates, the conjugated form of LC3(LC3-II) and SQSTM1/p62, are bothaccumulated; autophagosomes are alsopiled up. The LC3-II augmentation mightbe caused by either autophagy activationor a maturation block. These two possibi-lities could be distinguished by an auto-

phagy flux assay because treatment oflysosome inhibitors will further increaseLC3-II in the prior case but remainunchanged in the latter case. InTECPR1-deficient cells, the autophagyflux is blocked, suggesting that theautophagosome maturation is likelydefective without TECPR1.

LC3 tandemly fused with both GFPand mRFP is often employed to monitorautophagosome maturation. GFP, but notmRFP, is quenched in the acidic environ-ment, and therefore a lack of greenfluoresence serves as a marker for matureautophagosomes. Phagophores and auto-phagosomes decorated by LC3 displayboth green (GFP) and red (mRFP)fluorescence, while LC3-positive autolyso-somes are only labeled with red fluore-scence. In TECPR1 wild-type cells,red-only autolysosomes are dramaticallyincreased upon starvation, suggesting anincrease of mature autophagosomes.However, in TECPR1-deficient cells,nearly all the autophagic structures are

marked by both green and red signalsindicating accumulation of phagophoresand/or early autophagosomes. These

results further confirm that autophago-some maturation is blocked in the absenceof TECPR1.

Finally, we found the pleckstrin homo-logy (PH) domain of TECPR1, at theclose proximity of AIR, is critical for itsautophagic function. This PH domain

alone binds to PtdIns3P in vitro.However, the full-length TECPR1 dis-plays no PtdIns3P binding. Upon eitherbinding of ATG12ATG5 or deletion of

AIR, the full-length TECPR1 regains theability to interact with PtdIns3P. This isprobably because the ATG12ATG5interaction at the AIR region helps toexpose the PH domain of TECPR1,

which is otherwise concealed by AIR.Importantly, both full-length and AIR-deleted TECPR1 can rescue those auto-phagy phenotypes that we observed inTECPR1-deficient cells while PH-deletedTECPR1 could not. These results suggestthat the PtdIns3P binding activity ofTECPR1 is dependent on the association

with ATG12ATG5 and is essential for itsfunction in autophagosome maturation.

ATG12ATG5-ATG16 has long beenproposed to have a major role in phago-phore formation. This complex decoratesthe outer membrane of the formingautophagosome, where ATG12ATG5-

ATG16 recruits LC3 for its lipid conjuga-

tion. Interestingly, our study suggests that,in addition to its function in autophago-some formation, ATG12ATG5 may also

have an important role in autophagosomematuration by forming another complex

with TECPR1. Since ATG5 is essential forautophagosome formation, it is verydifficult to directly and specifically investi-gate its requirement for autophagosomematuration through either conventional

knockdown or knockout studies. Futureexperiments should be performed in an invitro system using purified recombinantproteins to further confirm this notion.

Autophagosome maturation is impli-cated in a wide spectrum of humandiseases. If this process goes wrong,damaged organelles, cytosolic proteinsand bacterial pathogens could bypasslysosomal degradation. Our study suggeststhat TECPR1 offers the fusion specificitybetween autophagosomes and lysosomesby interacting with ATG12ATG5 andPtdIns3P. Therefore, TECPR1 is likely anideal drug target, and dissecting itsregulation should provide new insightsinto the autophagosome maturation fieldand therapeutic tools for human diseases.

Acknowledgments

The work is supported by University ofCalifornia Cancer Research CoordinatingCommittee funds, a New Investigator

Award for Aging from the EllisonMedical Foundation, Hellman Family

Fund, American Cancer Society ResearchScholar Grant (RSG-11-274-01-CCG)and NIH R01 (CA133228) to Q.Z.

986 Autophagy Volume 8 Issue 6