Developmental Cell Article

TRIM Proteins Regulate and Can Target Autophagic Substrates by Direct Recognition

Michael A. Mandell,1 Ashish Jain,2 John Arko-Mensah,1 Santosh Chauhan,1 Tomonori Kimura,1 Christina Dinkins,1 Guido Silvestri,3 Jan Mu¨ nch,4 Frank Kirchhoff,4 Anne Simonsen,5 Yongjie Wei,6 Beth Levine,6 Terje Johansen,2 and Vojo Deretic1,* 1Department of Molecular and , University of New Mexico Health Sciences Center, 915 Camino de Salud NE, Albuquerque, NM 87131, USA 2Molecular Research Group, Institute of Medical Biology, University of Tromsø-The Arctic University of Norway, 9037 Tromsø, Norway 3Yerkes National Primate Research Center, Emory University, 3014 Yerkes, 954 Gatewood Road NE, Atlanta, GA 30329, USA 4Institute of Molecular Virology, Ulm University Medical Center, Meyerhofstrasse 1, 89081 Ulm, Germany 5Department of Biochemistry, Institute of Basic Medical Sciences, University of Oslo, 0317 Oslo, Norway 6Center for Autophagy Research and Howard Hughes Medical Institute, UT Southwestern Medical Center, 5323 Harry Hines Boulevard, Dallas, TX 75390, USA *Correspondence: [email protected] http://dx.doi.org/10.1016/j.devcel.2014.06.013

SUMMARY et al., 2011). ULK1 (Mizushima et al., 2011) and Beclin 1 (Liang et al., 1999) cooperate in the control of autophagy. A signaling Autophagy, a homeostatic process whereby eukary- cascade between the ULK1 and Beclin 1 systems has been es- otic cells target cytoplasmic cargo for degradation, tablished via an activating phosphorylation of Beclin 1 by ULK1 plays a broad role in health and disease states. Here (Russell et al., 2013). The subsequent stages of the pathway we screened the TRIM family for roles in autophagy are controlled by the mammalian paralogs of yeast Atg8. One and found that half of TRIMs modulated autophagy. of them, LC3B, is the most commonly used autophagosomal In mechanistic studies, we show that TRIMs asso- marker (Kabeya et al., 2000), whereas the role of other mamma- lian Atg8s is only beginning to be appreciated (von Muhlinen ciate with autophagy factors and act as platforms et al., 2012; Weidberg et al., 2010). At the end of a conjugation assembling ULK1 and Beclin 1 in their activated cascade initiated by Atg7, LC3s are lipidated at their C termini a states. Furthermore, TRIM5 acts as a selective auto- as a defining event in the building of autophagic membranes phagy receptor. Based on direct sequence-specific (Mizushima et al., 2011). A phagophore sequesters the captured recognition, TRIM5a delivered its cognate cytosolic cytoplasmic cargo destined for autophagic disposal, which re- target, a viral capsid protein, for autophagic degrada- quires fusion of the autophagosome with (Mizushima tion. Thus, our study establishes that TRIMs can et al., 2011). function both as regulators of autophagy and as auto- In contrast to bulk autophagy, targets of selective autophagy phagic cargo receptors, and reveals a basis for selec- are recognized by autophagy receptors (Johansen and Lamark, tive autophagy in mammalian cells. 2011; Kirkin et al., 2009a; Li et al., 2013; Thurston et al., 2012; von Muhlinen et al., 2010; Wild et al., 2011), including p62/sequesto- some 1 (p62) (Bjørkøy et al., 2005). The targets, earmarked by specific tags such as ubiquitin (Perrin et al., 2004) and INTRODUCTION (Thurston et al., 2012), are delivered via cognate receptors to nascent autophagosomes (Johansen and Lamark, 2011; Kirkin Autophagy is a eukaryotic homeostatic mechanism whereby et al., 2009b). In addition to placing ubiquitin tags on autophagic cells remove from their cytoplasm toxic aggregates, damaged targets (Huett et al., 2012), E3 ligases have been implicated in and surplus organelles, and invading pathogens or utilize bulk autophagy activation of key regulatory factors (Nazio et al., cytosol for metabolic needs (Mizushima et al., 2011). The key 2013) downstream of signaling from TLR4, a pattern recognition morphological presentation of autophagy is the appearance of receptor (PRR) (Shi and Kehrl, 2010). The engagement of PRRs in autophagosomes, the organelles that carry out cytoplasmic autophagy, as exemplified by TLR4 above, extends to nearly all cargo sequestration, driven by Atg factors (Mizushima et al., major classes of PRRs (Deretic et al., 2013). 2011; Yang and Klionsky, 2010). The canonical pathway leading TRIMs represent a large family of proteins typically consisting to formation in the cytosol of the autophagic isolation membrane of three motifs: an N-terminal RING domain, a B box, and a is under the control of the Ser/Thr protein kinase ULK1 (Atg1 in coiled-coil domain (Kawai and Akira, 2011). Additionally, most yeast), positioned downstream of mTOR and AMPK, which inte- TRIMs possess a variable C-terminal domain, which has a role grate nutritional and other signals (Mizushima et al., 2011). The in substrate binding (Kawai and Akira, 2011). Although TRIMs mTOR and AMP kinases phosphorylate ULK1, resulting in its represent a large family of PRRs (Jefferies et al., 2011; Kawai inactivation or activation, respectively (Egan et al., 2011; Kim and Akira, 2011; Ozato et al., 2008; Reymond et al., 2001), a

394 Developmental Cell 30, 394–409, August 25, 2014 ª2014 Elsevier Inc. Developmental Cell TRIMs Are Autophagic Receptors and Regulators

Figure 1. TRIM Proteins Regulate Autophagy (A) HeLa cells stably expressing mRFP-GFP-LC3B were subjected to TRIM knockdowns and treated with pp242, and high-content image analysis was performed using a Cellomics HCS scanner and iDEV software. Shown are images (epifluorescence) with nuclear stain (blue) and GFP signal (green). Top: nontargeting siRNA-transfected cells treated with carrier (DMSO) or pp242. White lines, cell borders; red, LC3B puncta borders. Bottom: representative images of cells subjected to knockdown of TRIM45 and TRIM2, both treated with pp242. (B) Average area of GFP-LC3B puncta per cell from cells treated as in (A) (data from multiple 96-well plates with identical siRNA arrangements represent means ± SE). Encircled are pp242-induced wells (right) and wells with vehicle controls DMSO (bottom left). TRIM knockdowns that reduced or increased LC3B puncta readout by 3 SDs (horizontal lines) from pp242-treated controls are indicated by corresponding TRIM numbers. Gray point (Bec), Beclin 1 knockdown; red point (numeral 5), TRIM5a. (C) Domain organization of TRIM subfamilies (I–XI; UC, unclassified). TRIM hits (LC3 puncta area >3 SDs ± cutoff) are listed on the right. (D) Representative images of TRIM knockdown cells under basal autophagy conditions. Scr, scrambled siRNA. (E) High-content image analysis (TRIM siRNA screen) under basal conditions (full medium). Encircled are scrambled siRNA controls: group on the left (filled diamonds), pp242-induced wells; group on the bottom right (open diamonds), DMSO vehicle. Bec, Beclin 1 knockdown. TRIM knockdowns with GFP-LC3 (legend continued on next page) Developmental Cell 30, 394–409, August 25, 2014 ª2014 Elsevier Inc. 395 Developmental Cell TRIMs Are Autophagic Receptors and Regulators

systematic analysis of their involvement in autophagy has not with the similarly mild effects in this assay reported for Atg6 been carried out. Because there are indications that TRIMs and Beclin 1 (Matsui et al., 2007; Suzuki et al., 2004). We next may be of relevance for autophagy (Barde et al., 2013; Khan mapped the TRIM5a-binding domain on p62 to the region et al., 2014; Niida et al., 2010; Pizon et al., 2013; Tomar et al., demarcated by residues 170–256 (Figures 2A and 2B). The 2012; Yang et al., 2013), here we tested the hypothesis that, biochemical analysis of p62-TRIM5a interaction was corrobo- akin to other PRRs, TRIMs may play a general role in autophagy. rated by immunofluorescence microscopy analysis, with p62 We performed a small interfering (si)RNA screen examining the and TRIM5a colocalizing in puncta that were heterogeneous in effects of TRIM knockdown on punctate LC3 and uncovered size and distribution (Figure S2A). that a large number of TRIMs affect autophagy. We show here Sequestosome 1/p62 plays a role in autophagy but also has that TRIMs interact with ULK1, Beclin 1, and mammalian other functions (Moscat and Diaz-Meco, 2009), and thus we Atg8s. Further, we show that TRIM5a (Reymond et al., 2001) tested whether TRIM5a connected with any additional auto- also acts as a receptor for selective autophagy. phagy factors. One of the best-defined motifs for interactions between autophagy factors is the LC3-interacting region (LIR). RESULTS Using the LIR consensus algorithm ([DEST]x(0,1)[WFY]{RKGP} {RKGP}[LIV]), which was defined based on alignment of 26 vali- TRIM Proteins Affect Autophagy dated LIR sequences and mutational analysis of the ULK1 and We employed a high-content image analysis (Figure 1A; Fig- ATG13 LIRs interacting with GABARAP (Alemu et al., 2012), we ure S1A available online) with the autophagosomal marker LC3 searched TRIM5a for the presence of putative LIRs. The best (Kabeya et al., 2000; Mizushima et al., 2010) to screen the effects match was the sequence 186-DFEQL-190. We tested TRIM5a on autophagy of TRIM knockdowns (Figures 1B and 1C; Figures interactions with the full complement of mammalian Atg8 paral- S1A and S1B). Two conditions were examined: autophagy ogs, of which LC3B is a member. LC3B showed minimal or no induced with the mTOR inhibitor pp242 (Figures 1A and 1B) signal in glutathione S-transferase (GST) pull-down experiments and basal autophagy (Figures 1D and 1E). Automated image with TRIM5a (Figure 2C). However, we detected robust interac- collection of >500 cells per siRNA was machine analyzed using tions in vitro with other mammalian Atg8 paralogs (mAtg8s) GA- preset scanning parameters and object mask definition (iDEV BARAP and GABARAPL1, and to a lesser extent with LC3A, software). Autophagy induction with pp242 resulted in a 17- LC3C, and GABARAPL2 (Figure 2C). Similar relationships with fold induction of GFP-LC3B puncta area. TRIMs whose mean a subset of the mAtg8s were seen in cells as determined by total area of GFP-LC3 per cell in three separate siRNA screen ex- coimmunoprecipitation experiments (Figure 2D). Whereas periments (autophagy induced with pp242) differed by >3 stan- LC3B signal in TRIM5a GST pull-down assays was negligible dard deviations either above or below the mean of pp242- (compared to the GST control; Figure 2C), the coimmunoprecipi- treated controls were reported as hits. Out of the 67 human tation studies in cell lysates suggested that LC3B may nonethe- TRIMs tested, knockdown of 21 different TRIMs reduced GFP- less be in protein complexes in vivo with TRIM5a (Figure 2D). LC3B puncta area (Figure 1B) or puncta numbers (Figure S1B) TRIM5a colocalized with punctate LC3B (Figures S2B and per cell under induced conditions to an extent comparable to S2C) and cofractionated with the membrane-associated LC3B- or exceeding the effect of Beclin 1 knockdown. Ten TRIMs II form (Figure S2D). A p62 knockdown reduced the levels of showed a converse effect. Additional TRIMs affected basal auto- LC3B in coimmunoprecipitates with TRIM5a (Figure S2E), sug- phagy (Figures 1D and 1E). Thus, a large fraction of TRIMs affect gesting that LC3B detected in vivo in TRIM5a complexes was autophagy. directly or indirectly affected by p62 levels. We next mapped the region of TRIM5a responsible for interactions with the TRIM5a Interacts with Sequestosome 1/p62 and mAtg8s LC3A and GABARAP through GST pull-down experi- Mammalian Atg8s ments (Figures 2E and 2F). We found that loss of the TRIM5a For detailed analysis of how a TRIM participates in autophagy, region encompassed by amino acids 103–347 ablated these in- we chose to study TRIM5a. The rationale for focusing initially teractions (Figure 2F). Thus, TRIM5a interacts directly with a on TRIM5a was threefold: (1) TRIM5a is physiologically highly subset of mAtg8s and p62. relevant in cell-autonomous retroviral restriction (Stremlau We next asked, using a subset of TRIMs from our screen, et al., 2004, 2006); (2) prior observations have indicated that whether other TRIMs interacted with p62 and mAtg8s. We TRIM5a may associate with the autophagy receptor p62 (O’Con- included representative TRIMs based on whether they modu- nor et al., 2010); and (3) despite association with p62, no connec- lated (TRIMs 17, 22, 49, and 55) or showed no apparent effect tions with autophagy have been previously suggested in the on (TRIMs 16 and 20) LC3B puncta. All TRIMs tested interacted removal of TRIM5a’s cognate target, the retroviral capsid protein with GABARAP (Figure 2G). TRIMs also interacted with LC3A p24. We first confirmed the effects of TRIM5a knockdowns on and p62 (Figure 2G), albeit with exceptions (TRIMs 16 and 20; LC3B puncta (Figures S1C–S1E) and LC3-II levels in response Figure 2G). Thus, binding to GABARAP is a common feature to autophagy induction (Figures S1F and S1G). Although the ef- among TRIMs, whereas other mAtg8s as well as p62 show var- fects on LC3-II conversion were modest, they were in keeping iable but still prominent association with the TRIMs tested. puncta area >3 SDs (horizontal bar) above unstimulated controls are indicated by corresponding TRIM numbers. Data represent means of two experiments. Numbers in squares, TRIMs scored as hits under both basal and induced conditions; circled numbers, hits scored only under basal conditions. Cells treated with TRIM63 siRNA showed signs of apoptosis and were excluded from consideration. See also Figure S1.

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Figure 2. TRIMs Interact with Sequestosome 1/p62 and Mammalian Atg8s (A and B) Mapping of the sequestosome 1/p62 region interacting with RhTRIM5a. (A) Domain organization of p62 and deletion/point mutation constructs employed to analyze interactions with TRIM5a as shown in (B). KIR, KEAP1-interacting region. (B) Myc-TRIM5a was radiolabeled with [35S]methionine by in vitro translation and analyzed by GST pull-down assays with GST-p62 fusion proteins. Top: autoradiogram of pull-down products. Bottom: Coomassie brilliant blue (CBB)-stained SDS-polyacrylamide gel with GST-p62 proteins. Note TRIM5a input in first lane. (C) GST pull-down analysis of interactions between radiolabeled TRIM5a and GST-tagged mammalian Atg8 paralogs. (D) Coimmunoprecipitation analysis of interactions between HA-TRIM5a and GFP-tagged mammalian Atg8s in lysates from cells expressing the indicated constructs. IP, immunoprecipitation; WB, western blot. (legend continued on next page)

Developmental Cell 30, 394–409, August 25, 2014 ª2014 Elsevier Inc. 397 Developmental Cell TRIMs Are Autophagic Receptors and Regulators

TRIMs Interact with ULK1, an Early Regulator of cipitated with TRIM5a (Figures 4A and 4B). In contrast, inactive Autophagy Initiation p-ULK1 (Ser-757) was disenriched in TRIM5a complexes (Fig- Overexpression of TRIM5a induced autophagy (Figures 3A and ures 4A and 4B). We also observed substantial colocalization 3B). In these experiments, we used TRIM5a clones from two between TRIM5a and p-ULK1 (Ser-317) (Figure 4C). Of note, different species, human (HuTRIM5a) and rhesus macaque p-ULK1 (Ser-317) displayed a punctate pattern (Figure 4C). (RhTRIM5a). Although HuTRIM5a and RhTRIM5a show differ- Thus, we considered a model in which TRIM5a affected cyto- ences associated with their binding to viral capsid proteins plasmic distribution of p-ULK1 (Ser-317). For this, we developed (Stremlau et al., 2006), overexpression of GFP-tagged TRIM5a a p-ULK1 assay (PULKA) based on high-content microscopy to from either source increased the abundance of LC3-II (Figure 3A). determine the abundance of p-ULK1 puncta. Using the PULKA This indicated that TRIM5a from either species could be used approach, we found that cells knocked down for TRIM5a had interchangeably in autophagy activation experiments. Using fewer p-ULK1 puncta (Figure 4D). This indicates that TRIM5a the standard bafilomycin A1 flux assay (Mizushima et al., may play a role in autophagy by concentrating active p-ULK1, 2010), we established that TRIM5a overexpression induced possibly at sites of autophagy initiation. autophagy rather than blocked autophagic maturation (Fig- ure 3A). Overexpression of GFP-TRIM5a also increased LC3 TRIMs Interact with the Autophagy Regulator Beclin 1 puncta relative to cells overexpressing GFP alone (Figure 3B; In addition to ULK1, autophagy initiation requires a second reg- Figure S3A) in an ATG7-dependent manner (Figure S3B). ulatory system centered on Beclin 1 (Liang et al., 1999; Mizush- Early autophagosomal structures in mammalian cells form in ima et al., 2011), which itself is a target of ULK1 (Russell et al., the vicinity of an endoplasmic reticulum-derived structure 2013). Thus, we considered whether TRIM5a action may engage termed the omegasome (Axe et al., 2008). We examined the Beclin 1. Beclin 1 coimmunoprecipitated with TRIM5a (Figures intracellular localization of TRIM5a relative to the omegasome 4E and 4F; Figure S4A). Furthermore, ATG14L (Itakura et al., marker DFCP1 (Figure 3C; Figure S3C). Stably transfected 2008; Sun et al., 2008) and AMBRA1 (Fimia et al., 2007), compo- HeLa cells expressing hemagglutinin (HA)-TRIM5a showed a nents of the Beclin 1 complex engaged in autophagic initiation, punctate cytoplasmic distribution of TRIM5a as previously were found in complexes with TRIM5a (Figures 4E and 4F; Fig- described (Reymond et al., 2001). TRIM5a showed morpholog- ure S4A). TRIM5a and Beclin 1 interaction was confirmed by ical linkage and spatial proximity with DFCP1 (Figure 3C). A proximity ligation assay (PLA; Figures S4B and S4C), which re- similar juxtaposition has previously been noted for DFCP1 and ports direct protein-protein interactions in situ (Figure S4C, the early autophagy factors of the ULK1 complex during charac- scheme) (So¨ derberg et al., 2006). The positive PLA results with terization of the mammalian autophagosome formation sites (Ita- Beclin 1-TRIM5a were comparable to those with proteins known kura and Mizushima, 2010). We thus tested whether TRIM5a to be in complexes with TRIM5a, namely p62 and TAB2 (O’Con- associated with ULK1 and found that TRIM5a and ULK1 colocal- nor et al., 2010; Pertel et al., 2011)(Figures S4B and S4C), but not ized (Figure 3D; Figure S3D). TAK1, which nevertheless colocalized with TRIM5a (Pertel et al., Based on their colocalization, we tested whether the two 2011)(Figure S4D). proteins interacted and found that HA-TRIM5a coimmunopreci- To map Beclin 1 domains required for interactions with pitated with GFP-ULK1 (Figure 3E) and endogenous ULK1 (Fig- TRIM5a, coimmunoprecipitation experiments were carried out ure S3E). We next mapped the ULK1-interacting region on with deletion mutants of Beclin 1. TRIM5a (HuTRIM5a and TRIM5a (Figures 3F and 3G; Figure S3F). Deletion mutants of RhTRIM5a) bound Beclin 1 at regions defined by residues TRIM5a lacking the C terminus of the protein (amino acids 1–255 (encompassing the Bcl-2 homology 3 and coiled-coil 347–493) lost association with Myc-ULK1 in coimmunoprecipi- domains) and 141–450 (encompassing the coiled-coil and evolu- tation studies, indicating that the SPRY domain of TRIM5a was tionarily conserved domains) (Figures 4G and 4H; Figure S4E). required for ULK1 inclusion in the complex (Figure 3G). When a The coiled-coil domain region (residues 141–265) was insuffi- panel of TRIMs that behaved similar to TRIM5a in the screen cient for TRIM5a binding (Figure 4H). Thus, TRIM5a directly in- was tested, they too coimmunoprecipitated with ULK1 (Fig- teracts with Beclin 1, likely at two sites (Figure 4G). We also map- ure 3H). One exception was TRIM55, a TRIM that lacks a ped the Beclin 1-interacting region on TRIM5a (Figures 5A and SPRY domain. These findings indicate that TRIM5a and addi- 5B). Only the TRIM5a deletion mutant lacking the amino acids tional TRIMs act early in the autophagy pathway and that TRIMs’ 104–493 (consisting of part of the B box domain and all of the SPRY domains are required to engage ULK1. coiled-coil and SPRY domains) lost the ability to interact with Be- clin 1, whereas all other TRIM5a deletion mutants retained Beclin TRIM5a Interacts with Activated ULK1 and Affects Its 1 binding (Figure 5A). These data suggest the presence of two Intracellular Distribution separate TRIM5a-interacting regions on Beclin 1 and a longer ULK1 activity is regulated by its phosphorylation status (Egan TRIM5a region required for interactions with Beclin 1. et al., 2011; Kim et al., 2011), with phosphorylations at Ser-317 We tested other TRIMs (TRIMs 6, 22, 49, and 55) for Beclin 1 activating and at Ser-757 inactivating ULK1 (Kim et al., 2011). binding. As was the case for ULK1, all TRIMs tested bound to We found that active phospho(p)-ULK1 (Ser-317) coimmunopre- Beclin 1 with the exception of TRIM55 (Figure 5C). These findings

(E) Domain organization of TRIM5a and schematic of deletion mutants used for mapping experiments in (F). Dotted lines denote deleted regions of the protein. (F) Analysis of TRIM5a domains interacting with LC3A and GABARAP. (G) GST pull-down analysis of binding between the indicated radiolabeled TRIM proteins and GST-LC3A, GST-GABARAP, and GST-p62. See also Figure S2.

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Figure 3. TRIM5a and Additional TRIMs Interact with ULK1 (A) Top: 293T cells were transfected with GFP-tagged human (HuT5a) or rhesus (RhT5a) TRIM5a or GFP alone and treated or not with bafilomycin A1 (Baf), and levels of LC3B and actin were assayed by immunoblot. Bottom: quantitation of LC3B-II:actin ratios. CT, control without Baf. (B) High-content analysis of endogenous LC3 puncta in HeLa cells (full media) transfected with GFP or GFP-TRIM5a. Object masks: white contour line, gating for primary objects (GFP-positive cells). White internal small object masks, LC3B puncta. White asterisks, GFP-negative cells (manually entered; excluded from analysis by iDEV software). Graph: quantification of LC3B puncta area per green fluorescent (GFP+ or GFP-TRIM5a+) cell. (legend continued on next page) Developmental Cell 30, 394–409, August 25, 2014 ª2014 Elsevier Inc. 399 Developmental Cell TRIMs Are Autophagic Receptors and Regulators

indicate that TRIM5a, as well as additional SPRY domain-con- Thus, the tested SPRY-endowed TRIMs acted as platforms for taining TRIMs that behaved similar to TRIM5a in the screen, in- the assembly of autophagy regulators ULK1 and Beclin 1 into teracted with Beclin 1, another key regulator of autophagy. joint complexes.

a TRIM5 Activates Beclin 1 TRIM17 Focuses Autophagic Machinery to a Localized a We next tested whether TRIM5 affected Beclin 1 activation Domain in the Cell states. Beclin 1 is negatively regulated by two principal inhibitory A knockdown of TRIM17 in the siRNA screen increased LC3 binding partners, Bcl-2 (Wei et al., 2008) and TAB2 (Criollo et al., puncta under both inducing and basal conditions. Accordingly, 2011; Takaesu et al., 2012). A dissociation of these factors is overexpression of TRIM17 reduced the number of LC3B puncta necessary to initiate autophagy (Criollo et al., 2011; Takaesu per cell (Figure 6A). When we examined these cells for TRIM17 a et al., 2012; Wei et al., 2008). Overexpression of TRIM5 (Hu- localization, it became evident that TRIM17 formed one to two a a TRIM5 or RhTRIM5 ) caused a dissociation of Bcl-2 from prominent large structures (PLSs) per cell (Figure 6A), observed Beclin 1, as evidenced by diminished amounts of Bcl-2 that also with an antibody against endogenous TRIM17 (Figure S6A). coimmunoprecipitated with Beclin 1 (Figure 5D). Overexpression Endogenous LC3B puncta coalesced with TRIM17 PLS profiles in a of TRIM5 also caused dissociation of Beclin 1 from TAB2, evi- GFP-TRIM17-overexpressing cells (Figures 6A and 6B). Similarly, denced by lower amounts of Beclin 1 found in TAB2 complexes DFCP1 puncta, indicators of active autophagosome formation (Figure 5E). We confirmed this using PLA methodology, and (Axe et al., 2008), associated with TRIM17 PLSs (Figure 6C). a observed TRIM5 -dependent reduction in the PLA signal repre- TRIM17 bound ULK1 and Beclin 1 (Figures S6B and S6C) and senting Beclin 1-TAB2 interactions when cells expressing GFP- furthermore enriched ULK1 in Beclin 1 complexes (Figure 5G), a TRIM5 were compared to cells expressing GFP (Figure S5A). just like most other TRIMs acting as inducers of autophagy. In a Thus, TRIM5 derepresses Beclin 1 by causing release of its cells with overexpressed TRIM17, p-ULK1 dots were reduced negative regulators. in total area per cell (Figure S6D) and changed their normal pattern of dispersed small puncta (<1.3 mm) within the cytoplasm to coa- TRIMs Serve as Platforms to Assemble ULK1 and Beclin lescence at the PLS (>3 mm) (Figures 6D and 6E). In contrast, 1 into a Complex neither TRIM5a (Figures S6E and S6F) nor TRIM22 (Figures 6D Because TRIM5a binds both ULK1 and Beclin 1, we tested the and 6E) overexpression caused this p-ULK1 phenotype. The possibility that it could act as a platform upon which Beclin 1 unique features of TRIM17 in organizing active autophagic and ULK1 assembled together. ULK1 and Beclin 1 coimmuno- machinery at the PLS also extended to its recruitment of Exo84, precipitated in cells expressing GFP-TRIM5a but not GFP alone an exocyst component previously associated with active auto- (Figure 5F). Furthermore, we detected the form of Beclin 1 phagic complexes, to ULK1 complexes (Bodemann et al., 2011), phosphorylated by ULK1 (phospho-Beclin 1 at Ser-15; Russell contrasting with TRIM5a (Figure S6G). Thus, TRIM17 organizes et al., 2013) in complexes with TRIM5a (Figure S5B). Thus, active autophagy complexes but focuses them at the PLS. TRIM5a acts as a platform for ULK1 and Beclin 1 assembly The above analysis indicates that although TRIM17 appeared and activation. as a negative regulator of autophagy in the initial siRNA screen, We next tested whether the TRIM5a capacity for bringing its effect on LC3 puncta distribution is due to the recruitment of together ULK1 and Beclin 1 extended to other TRIMs (Figure 5G). autophagic factors to a localized, limited area of the cell. We next We included the same TRIM panel (TRIMs 6, 22, 49, and 55) asked whether and how other TRIMs can overcome this effect. employed for the analysis of binding of ULK1 or Beclin 1, and When TRIM5a and TRIM17 were coexpressed in HeLa cells, found that those TRIMs (TRIMs 6, 22, and 49) that bound TRIM5a exerted dominance over TRIM17 (Figure 6F) by altering ULK1 or Beclin 1 individually also brought these molecules the localization pattern of ULK1 and TRIM17 away from forming together into a complex. Hence, coexpression of these TRIMs a PLS and toward a dispersed cytoplasmic overall distribution enriched ULK1 in the immunoprecipitated Beclin 1 complexes. that is typical of TRIM5a. Moreover, TRIM17 and ULK1 now co- In contrast, TRIM55, which lacks a SPRY domain and does not localized strongly with TRIM5a (Figure 6F). TRIM22 showed associate with either ULK1 or Beclin 1, had no such effect. the same effect, acting dominantly over TRIM17 (Figure 6F).

Data represent means ± SE; n R 3 experiments; *p < 0.05 (t test). (C) Confocal microscopy of HeLa cells stably expressing HA-TRIM5a and transiently expressing GFP-DFCP1. Numeral 1, a punctum displayed in line-tracing profile below. Arrows indicate puncta showing the juxtaposition of DFCP1 and TRIM5a. Dotted-line insets contain zoomed-in regions shown enlarged in the solid- line insets. (D) Confocal microscopy analysis of HA-tagged TRIM5a (green) and endogenous ULK1 (red) in HeLa cells. Numeral 2, two puncta displayed in line-tracing profile below. Arrows indicate puncta showing colocalization between ULK1 and TRIM5a. Dotted-line insets contain zoomed-in regions shown enlarged in the solid-line insets. (E) Lysates from HeLa cells stably expressing HA-tagged TRIM5a and transiently overexpressing either GFP-ULK1 or GFP alone were subjected to immuno- precipitation with anti-HA, and immunoblots were probed with anti-GFP. (F) Domain organization of TRIM5a and deletion constructs used in mapping experiments. Dotted lines denote deleted regions of the protein. (G) Coimmunoprecipitation analysis of full-length or deletion variants of TRIM5a (as GFP fusions; asterisks denote fusion products on the bottom blot) with Myc-ULK1 transiently expressed in 293T cells. (H) Coimmunoprecipitation analysis of interactions between the indicated TRIMs (as GFP fusions) and Myc-ULK1 in 293T cell extracts. See also Figure S3.

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Figure 4. TRIM5a Interacts with Activated ULK1 and with Beclin 1 (A) Lysates from HeLa cells stably expressing HA-TRIM5a and transiently transfected with GFP-ULK1 were immunoprecipitated with anti-HA and blots were probed with antibodies against Ser-317 or Ser-757 phospho-ULK1. (B) Lysates from HeLa cells stably expressing HA-TRIM5a were immunoprecipitated with anti-HA, and blots were probed as in (A) for endogenous ULK1. Graph: ratio of immunoprecipitated phospho-ULK1 to phospho-ULK1 in the input. (C) Confocal microscopy of cells stably expressing HA-tagged TRIM5a (green) and stained to detect p-ULK1 (p-Ser-317; red). Arrows indicate puncta showing colocalization between p-ULK1 and TRIM5a. Dotted-line insets contain zoomed-in regions shown enlarged in the solid-line insets. The scale bar represents 2 mm. (D) High-content analysis of endogenous p-ULK1 (p-Ser-317; red) in control HeLa cells or cells subjected to TRIM5a knockdown. Blue, nuclei. Graph: area of phospho-ULK1 (Ser-317) puncta per cell. The scale bar represents 5 mm. (legend continued on next page) Developmental Cell 30, 394–409, August 25, 2014 ª2014 Elsevier Inc. 401 Developmental Cell TRIMs Are Autophagic Receptors and Regulators

Thus, the codistribution of autophagic machinery with various recognizes the HIV-1 capsid but is unable to recognize the equiv- TRIMs in the cell determines the localization of autophagosome alent simian immunodeficiency virus (SIV) capsid (Stremlau et al., formation. 2004, 2006). A prediction based on this property of RhTRIM5a is that rhesus TRIM5a and autophagy can act upon HIV but cannot A Role for TRIM5a as an Autophagic Receptor affect SIV. To test this hypothesis, we employed an assay with The above studies indicate a regulatory role for TRIMs in auto- viral measured by luciferase outputs. Autophagy, phagy. Because TRIM5a is known to bind to a retroviral capsid induced by starvation, as in the p24 assays above, resulted in as a part of its antiretroviral activity (Stremlau et al., 2006), we a reduced output of virally encoded luciferase only with HIV wondered whether TRIM5a plays an additional role in autophagy but not with SIV (Figures 7E and 7F). Moreover, Atg7, Beclin 1, by targeting its cognate cargo for autophagic degradation. The p62, and TRIM5a were all required for optimal effects, again RhTRIM5a SPRY domain binds to the HIV-1 capsid composed affecting luciferase outputs only in the case of HIV but not SIV of the viral protein p24 (Stremlau et al., 2006)(Figure S7A). We (Figures 7E and 7F). These studies establish that mobilization thus tested whether the HIV-1 capsid protein p24 can be a sub- of the viral target for degradation by the autophagic apparatus strate for lysosomal degradation in rhesus cells, FRhK4, which directly correlates with the target’s amino acid sequence recog- express endogenous RhTRIM5a. The experiments were carried nized by TRIM5a. Collectively, these findings establish TRIM5a out using vesicular stomatitis virus G protein (VSVG)-pseudo- as an autophagic receptor. typed HIV-1 core viral particles. The experiments in Figures S7B and S7C indicated that inhibition of lysosomal proteases Interaction of TRIM5a with mAtg8s Is Necessary for Its protected HIV-1 p24 from degradation in rhesus cells. We next Action as an Autophagic Regulator and a Receptor tested whether the observed lysosomal degradation of p24 As established in the previous sections, TRIM5a interacts with was through autophagy. The data in Figures 7A and 7B indicated mAtg8s, with the most prominent interaction being with GA- that p24 degradation was dependent on the autophagy factors BARAP. We used GABARAP association with TRIM5a to delimit Atg7, Beclin 1, p62, and TRIM5a. Induction of autophagy by star- the sequence required for binding (Figures 7G and 7H). A 20-mer vation increased degradation of p24 in control cells but not in peptide array representing the entire TRIM5a sequence in incre- FRhK4 cells subjected to Atg7, Beclin 1, p62, or TRIM5a knock- ments staggered by 3-amino acid residues was subjected to a downs (Figures 7A and 7B). Furthermore, ALFY/WDFY3, a dot blot with GST-GABARAP as a probe (Figure 7G). Several potentiator of p62-dependent autophagy (Filimonenko et al., peptides showed positive signals. We focused on the positive 2010), colocalized with TRIM5a (Figure S7D), and a knockdown peptides with at least three consecutive binding signals. One of ALFY in FRhK4 cells protected p24 from degradation, abro- such series containing exclusively the canonical LIR sequence gating the effect of starvation (Figures S7E and S7F). Collec- 186-DFEQL-190 (Figure 7G, dashed-framed dots) conferred tively, these data are consistent with the interpretation that p24 only a weak GST-GABARAP binding. However, those peptides is a target for autophagic degradation and that this process is (Figure 7G, solid line-framed dots) that contained an adjacent directed by TRIM5a in cooperation with p62 and ALFY. A phys- sequence, WEESN, located at positions 196–200, conferred iological relevance of the autophagic action of TRIM5a was indi- strong GABARAP binding. Mutational analysis of key residues cated by the role that TRIM5a and autophagic factors played in (LIR-1*: AEQA, instead of FEQL; LIR-2*: AAESN, instead of diminishing the p24 levels in rhesus primary CD4+ T cells upon WEESN; Figure 7I; Figure S7I) indicated that GABARAP and infection (Figures 7C and 7D). other mAtg8s tested utilized both sequences. These experi- Because upon viral entry TRIM5a recognizes the capsid pro- ments identified LIR-1 and LIR-2 in TRIM5a as the key motifs in- tein only in the specific tertiary structure of the viral capsid teracting with mammalian Atg8s, with LIR-2 showing similarity to (Stremlau et al., 2006), we considered for further study the use LIR variants reported to bind strongly to GABARAP (Knævelsrud of a viral infection output assay. This was possible, because et al., 2013; Ma et al., 2013). knocking down TRIM5a or autophagy factors resulted in an in- We employed the above information to test whether crease of the abundance of proviral DNA (Figure S7G) and TRIM5a’s interactions with mAtg8s were important for its auto- reverse transcriptase (Figure S7H), in accordance with the re- phagic functions. RhTRIM5a mutant with altered LIR-1 and LIR- sults from the p24 assay described above. Having established 2 motifs (RhTRIM5a LIR-1*&2*) showed diffuse cytoplasmic that autophagy can lead to elimination of a portion of the appearance instead of punctate distribution (Figure 7J; Fig- incoming HIV-1 viral particles in cells expressing RhTRIM5a, ure S7J). Whereas expression of the wild-type RhTRIM5a re- we next sought to determine whether this function was depen- sulted in increased p-ULK1 puncta (Ser-317), mutation of dent on the specific interaction between TRIM5a as an autopha- LIR-1 and LIR-2 abrogated this effect (Figure 7K; Figure S7K). gic receptor and its viral protein target. To do this, we utilized a RhTRIM5a’s ability to increase LC3B puncta, as a readout of well-characterized feature of the RhTRIM5a SPRY domain that autophagy induction, was abrogated by LIR-1 and LIR-2

(E) Top: lysates from cells stably expressing HA-tagged TRIM5a were immunoprecipitated with anti-HA, and immunoblots were probed as indicated. Bottom: lysates as above were subjected to immunoprecipitation with anti-Beclin 1, and immunoblots were probed for HA-TRIM5a. (F) Lysates from rhesus epithelial cells (FRhK4) were immunoprecipitated with anti-Beclin 1, and immunoblots were probed with the indicated antisera. (G and H) Mapping of Beclin 1 regions interacting with TRIM5a. Schematic (G) of Beclin 1 constructs used in immunoprecipitation experiments in (H). Lysates of 293T cells coexpressing GFP-tagged TRIM5a and the indicated FLAG-tagged Beclin 1 constructs in (D) were immunoprecipitated with anti-GFP, and immu- noblots were probed as indicated. BH, Bcl-2 homology; CCD, coiled-coil domain; ECD, evolutionarily conserved domain. Data represent means ± SE; n R 3 experiments; *p < 0.05 (t test). See also Figure S4.

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Figure 5. TRIM5a Interacts with Activated Beclin 1 and Assembles ULK1 with Beclin 1 (A) Domain organization of TRIM5a and deletion constructs used in mapping experiments in (B). Dotted lines denote deleted regions of the protein. (B) Coimmunoprecipitation analysis of full-length or deletion mutants of GFP-TRIM5a with FLAG-tagged Beclin 1. (C) Coimmunoprecipitation analysis of interactions between the indicated TRIMs (as GFP fusions) and FLAG-Beclin 1 in 293T cell extracts. (D) Bcl-2-Beclin 1 complexes assessed by coimmunoprecipitation from control cells or cells expressing HA-HuTRIM5a or HA-RhTRIM5a. (E) Abundance of TAB2-Beclin 1 complexes assessed by coimmunoprecipitation from 293T cells expressing GFP-TRIM5a or GFP alone. Transfected cells were subjected to TAB2 immunoprecipitation, and intensities of Beclin 1 bands in the precipitates were normalized to TAB2 in the same samples. Differences in the normalized values (set at 100% for GFP-expressing cells) were assessed between cells expressing GFP or GFP-TRIM5a. Data represent means ± SE; n = 3 experiments; *p < 0.05 (t test). (F) Assessment of TRIM5a effects on ULK1 presence in Beclin 1 complexes. 293T cells were transiently transfected with Myc-ULK1, FLAG-Beclin 1, and either GFP-TRIM5a or GFP alone. Lysates were immunoprecipitated with anti-FLAG, and immunoblots were probed as indicated. (G) Lysates from 293T cells expressing GFP or the indicated GFP-TRIMs, Myc-ULK1, and FLAG-Beclin 1 were treated as in (F). See also Figure S5. mutations (Figure 7L; Figure S7L). Mutations of LIR-1 and LIR- ure 7M). Thus, LIR-1 and LIR-2, the motifs in TRIM5a, are 2, which caused the above phenotypes, also precluded essential for its regulatory effects on autophagy and for its abil- RhTRIM5a from targeting HIV-1 p24 for degradation (Fig- ity to act as an autophagic receptor.

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Figure 6. TRIM17 Focuses Active Autophagy Machinery, whereas TRIM5a and TRIM22 Can Redirect Its Localization (A) Effect of GFP-TRIM17 overexpression on LC3B puncta abundance in HeLa cells. Representative confocal micrographs showing GFP-TRIM17 and LC3B (red) under starvation conditions. PLS, prominent large structure containing GFP-TRIM17. Graph: number of LC3B puncta per cell. Data represent means ± SEM; n R 30 cells; *p < 0.05 (t test). (B) Confocal micrographs showing the association between the TRIM17 PLS profile (green) and endogenous LC3B (red) in HeLa cells under starvation conditions. The scale bars represent 2 mm. (C) Confocal micrograph of the association between the PLS (labeled with mCherry-TRIM17; red) and omegasome marker GFP-DFCP1. (D) Intracellular distribution of endogenous p-ULK1 (p-Ser-317) in HeLa cells expressing GFP-TRIM17 or GFP-TRIM22. Dotted-line insets contain zoomed-in regions shown enlarged in the solid-line insets.

(legend continued on next page)

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DISCUSSION 2001), this may differentially regulate levels of individual TRIMs and positioning of TRIMosomes in selective autophagy. This study shows that TRIM proteins as a family broadly affect In its role as an autophagic cargo receptor, TRIM5a directly autophagy. The interactions between autophagy factors and recognizes viral capsid sequences via its SPRY domain (Strem- TRIMs (e.g., TRIMs 5a, 6, 16, 17, 20, 22, 49, and 55) are extensive lau et al., 2004, 2006). This is an example of selective autophagy and involve both regulators and effectors (ULK1, Beclin 1, in mammalian cells, which we propose occurs via direct sub- mAtg8s, and p62/sequestosome 1). In their regulatory role, strate recognition by TRIMs. We note that TRIMs contain TRIMs 5a, 6, 17, 22, and 49 act as platforms to bring together SPRY or other types of C-terminal domains with the potential ULK1 and Beclin 1 into a single complex. By affecting the distri- to recognize diverse protein targets or other molecular patterns bution of phospho-ULK1 (p-Ser-317), these TRIMs position the (Kawai and Akira, 2011): COS, microtubule binding; FN3, DNA autophagy machinery. Our data with TRIM5a also reveal that or heparin binding; PHD, histone binding; BROMO, acetylated TRIMs can play the role of cargo receptors for selective auto- Lys residue binding; FIL, actin crosslinking; and NHL, protein in- phagy: TRIM5a targets a retroviral capsid protein for autophagic teractions. Thus, we propose that TRIM proteins, as a group, degradation by direct recognition of the target’s amino acid comprise a class of broad-repertoire high-fidelity selective auto- sequence in a manner dependent on interactions with mAtg8s. phagic receptors. These receptors may directly recognize their Based on these features, we propose a model in which TRIMs cognate targets without a need for ubiquitin tagging, which embody within one entity several aspects of selective autophagy is the currently prevailing model of how autophagy targets its such as target recognition, recruitment of the autophagy initia- cargo (Shaid et al., 2013). The receptor function of TRIM5a tion machinery, and execution of the autophagic degradation shown here is reminiscent of selective autophagy in yeast, where of the cargo. this process is independent of ubiquitin tags, including the Cvt Our screen reveals a broad engagement of TRIMs in auto- pathway (Lynch-Day and Klionsky, 2010), mitophagy (Kanki phagy. Additional work supports TRIM connections with auto- et al., 2009; Okamoto et al., 2009), and pexophagy (Farre´ phagy: TRIM13 overexpression can induce autophagy (Tomar et al., 2008). Should other TRIMs act as receptors that directly et al., 2012); TRIM28 may regulate autophagy (Barde et al., bind to their targets via cargo-recognition domains, this would 2013; Yang et al., 2013); several TRIMs interact with p62 (Fusco confer exclusivity of autophagic targeting that a generic tagging et al., 2012; Khan et al., 2014; Kim and Ozato, 2009; Pizon et al., with ubiquitin lacks. 2013; Tomar et al., 2012); the expression of TRIM55 correlates In conclusion, our study reports the recognition of a global with that of autophagy factors (Perera et al., 2011; Pizon et al., control of autophagy by TRIMs. TRIM proteins interact with com- 2013); TRIMs are on lists in genome-wide autophagy screens ponents of the autophagic apparatus including mAtg8s, p62, (Behrends et al., 2010; Lipinski et al., 2010; McKnight et al., and activated ULK1 and Beclin 1. TRIM5a acts both as a regu- 2012); and TRIM21 and murine TRIM30a induce lysosomal lator of autophagy by providing a platform for the assembly of degradation of certain targets (Niida et al., 2010; Shi et al., activated ULK1 and Beclin 1 and as a receptor for selective auto- 2008). phagy. Thus, TRIM5a embodies in one core entity two essential The assembly of autophagic machinery (ULK1, Beclin 1, and aspects of selective autophagy—recognition of the cargo and mAtg8s) on TRIMs leads us to propose the concept of the initiation of autophagy. Because TRIMs play diverse physiolog- TRIMosome (Figure S7M). The TRIMosome acts as a platform ical roles (Barde et al., 2013; Cavalieri et al., 2011; Chen et al., to focus selective autophagy on highly specific targets. How- 2012; Jefferies et al., 2011; Kawai and Akira, 2011; Ozato ever, additional processes may also be at play. For instance, et al., 2008; Pertel et al., 2011; Versteeg et al., 2013) and have TRIM28 was reported to influence expression of autophagy been linked to human inflammatory diseases and cancer (Hata- genes via microRNAs (Barde et al., 2013). Furthermore, keyama, 2011; Jefferies et al., 2011; Kawai and Akira, 2011), our TRIM55 in our experiments did not bind ULK1 and Beclin 1 study invites explorations of the broad connections revealed (albeit it bound mAtg8s) but still strongly affected autophagy. here between TRIMs and autophagy. TRIM17 and TRIM22, although both binding to ULK1 and Be- clin 1, differently distribute the activated form of ULK1 (p-Ser- EXPERIMENTAL PROCEDURES 317) in the cell, possibly to accomplish differential physiological tasks or to capture different cognate cargoes. TRIM17 focuses See also Supplemental Experimental Procedures. active autophagic machinery in a very restricted part of the cell, of as yet to be determined function. This can explain why TRIM Family Screen in the siRNA screen TRIM17 knockdown increased generic HeLa cells stably expressing mRFP-GFP-LC3B were cultured in 96-well plates LC3 puncta as a readout of nonselective (pp242-induced) auto- containing SMARTpool siRNAs and transfection reagent (Dharmacon). Forty- eight hours after plating, cells were treated with pp242 (as inducer of auto- phagy. In competition experiments, TRIM5a and TRIM22 both phagy) or DMSO (control) for 2 hr, fixed, and stained with Hoechst 33342. redirected and dispersed the autophagic machinery from the Plates with cells were subjected to high-content analysis for image acquisition TRIM17 foci. Because TRIM expression responds to cytokines and data processing. Three separate siRNA screens for induced autophagy (Carthagena et al., 2009) and other signals (Bodine et al., and two separate siRNA screens for basal autophagy were carried out with

(E) Fluorescence intensity line tracings show phospho-ULK1 localization relative to GFP-tagged TRIMs. (F) Confocal micrograph of HeLa cells coexpressing mCherry-TRIM17 with Myc-ULK1 and GFP-TRIM5a (top) or with Myc-ULK1 and GFP-TRIM22 (bottom). The scale bars represent 5 mm. See also Figure S6.

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Figure 7. Autophagy Degrades the Protein Target of TRIM5a in a Manner Requiring Direct Cargo Recognition and mAtg8-Interacting Motifs in TRIM5a (A and B) Levels of intracellular p24 were determined by immunoblotting lysates from rhesus cells (FRhK4) that had been subjected to knockdown of autophagy factors (Scr, scrambled siRNA) or TRIM5a and then exposed to pseudotyped HIV-1 for 4 hr under full media or starvation conditions. (C and D) Immunoblot-based assessment of HIV-1 p24 in primary rhesus CD4+ T cells subjected to the indicated knockdowns, infected with VSVG-pseudotyped HIV-1, and induced for autophagy by starvation for 4 hr. (E and F) Luciferase activity of fed or starved FRhK4 cells subjected to the indicated knockdowns and infected with luciferase-expressing pseudotyped HIV-1

(E) or SIVmac239 (F). (G and H) Binding of GST-GABARAP to a TRIM5a peptide array. A 20-mer peptide array corresponding to the entire TRIM5a sequence scanned in 3-amino acid residue shifts was subjected to a dot blot with GST-GABARAP as a probe. (legend continued on next page)

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the same cutoff (>3 SDs change relative to the mean of stimulated or unstimu- were generated by cotransfection of plasmids encoding VSVG protein and lated controls, respectively) for hits. the NL43 or SIVmac239 clones lacking the env gene into 293T cells. Rhesus blood was collected under protocols approved by the Emory University Insti- High-Content Image Analysis tutional Animal Care and Use Committee. High-content image analysis was performed using a Cellomics HCS scanner and iDEV software (Thermo Scientific). Automated epifluorescence image SUPPLEMENTAL INFORMATION collection was carried out until a minimum of 500 cells per well per siRNA knockdown per plate was acquired. Epifluorescence images were machine Supplemental Information includes Supplemental Experimental Procedures analyzed using preset scanning parameters and object mask definitions. Cells and seven figures and can be found with this article online at http://dx.doi. were identified based on nuclear staining and cell outlines defined by back- org/10.1016/j.devcel.2014.06.013. ground staining of the cytoplasm, and the mean per cell total area of GFP puncta or number of GFP puncta per cell was reported. Autophagy induction with pp242 resulted in a Z0 value of 0.52. When results were expressed as ACKNOWLEDGMENTS puncta area per cell, the units corresponded to mm2/cell. We thank George Kyei, Manohar Pilli, Nicolas Dupont, Eliseo Castillo, Steven High-Content Analysis in Subpopulations of Transfected Cells Bradfute, Michal Mudd, and J.L. Guan. This work was supported by grants HeLa cells were transfected with GFP or GFP-TRIM5a plasmids with or AI042999 and AI111935 from the NIH and a grant from the Bill and Melinda without siRNA and cultured in full media for 48 hr. Cells were then immuno- Gates Foundation, and in part by the National Center for Research Resources stained to detect endogenous LC3. High-content image analysis was used and the National Center for Advancing Translational Sciences of the NIH in a mode discriminating transfected (GFP-positive cells) from untransfected through grant UL1 TR000041. M.A.M. and C.D. were supported by NIH training cell subpopulations. High-content image analysis, discriminating transfected grant T32AI007538. T.K. was supported by Manpei Suzuki Founda- cell subpopulations, was performed using a Cellomics HCS scanner and tion. F.K. acknowledges the German Research Foundation and the Zeiss iDEV software (Thermo Scientific); >200 cells were analyzed per treatment in Foundation. G.S. was supported by NIH grant P51OD011132 to the Yerkes quadruplicate per experiment. Cell outlines were automatically determined National Primate Research Center of Emory University. B.L. was supported based on background nuclear staining, and the mean total area of punctate by NIH grant CA109618 and Cancer Prevention and Research Institute of LC3, p-ULK1 (Ser-317), or TRIM5a per cell was determined within the subpop- Texas grant RP120718-P1. T.J. was supported by grant 196898 from the Nor- ulation of cells that were successfully transfected as determined by having wegian Research Council and grant 71043-PR-2006-0320 from the Norwegian above-background GFP fluorescence. Cancer Society.

Confocal Microscopy Received: February 6, 2014 Fluorescence confocal microscopy was carried out using a Zeiss META Revised: May 11, 2014 microscope. Accepted: June 17, 2014 Published: August 7, 2014 Antibodies for Immunoblotting, Immunolabeling for Microscopy, and Coimmunoprecipitation REFERENCES Antibodies used were: ATG7 (Santa Cruz), ATG14L (MBL), Beclin 1 (Novus and Santa Cruz), Flag (Sigma), HA (Sigma and Roche), p62 (Abcam), TAB2 (Santa Alemu, E.A., Lamark, T., Torgersen, K.M., Birgisdottir, A.B., Larsen, K.B., Jain, Cruz), TAK1 (Abcam), TRIM5a (Abcam), ULK1 (Sigma), ULK1 p-Ser-317 and p- A., Olsvik, H., Øvervatn, A., Kirkin, V., and Johansen, T. (2012). ATG8 family Ser-757 (Cell Signaling), Beclin 1 p-Ser-15 (Abbiotec), FIP200 (provided by J.L. proteins act as scaffolds for assembly of the ULK complex: sequence require- Guan, University of Cincinnati), Exo84 (Abcam), and c-Myc (Santa Cruz). All ments for LC3-interacting region (LIR) motifs. J. Biol. Chem. 287, 39275– other antibodies and methods are described in Supplemental Experimental 39290. Procedures. Axe, E.L., Walker, S.A., Manifava, M., Chandra, P., Roderick, H.L., Habermann, A., Griffiths, G., and Ktistakis, N.T. (2008). Autophagosome Peptide Array Overlay Assay and GST Pull-Down Assay formation from membrane compartments enriched in phosphatidylinositol Peptide arrays were synthesized on cellulose membranes using a MultiPep 3-phosphate and dynamically connected to the endoplasmic reticulum. automated peptide synthesizer (INTAVIS Bioanalytical Instruments). Peptide J. Cell Biol. 182, 685–701. interactions were probed by overlaying the membranes with 1 mg/ml recombi- Barde, I., Rauwel, B., Marin-Florez, R.M., Corsinotti, A., Laurenti, E., Verp, S., nant protein for 2 hr. Bound proteins were detected with horseradish peroxi- Offner, S., Marquis, J., Kapopoulou, A., Vanicek, J., and Trono, D. (2013). A dase-conjugated anti-GST antibody (1:5,000; clone RPN1236; GE Health- KRAB/KAP1-miRNA cascade regulates erythropoiesis through stage-specific care). GST pull-down assays are described in Supplemental Experimental control of mitophagy. Science 340, 350–353. Procedures. Behrends, C., Sowa, M.E., Gygi, S.P., and Harper, J.W. (2010). Network orga- Viral Infection nization of the human autophagy system. Nature 466, 68–76. Viral detection and yield measurements are detailed in Supplemental Experi- Bjørkøy, G., Lamark, T., Brech, A., Outzen, H., Perander, M., Overvatn, A., mental Procedures. HIV-1 or SIVmac239 viruses for single-cycle infection Stenmark, H., and Johansen, T. (2005). p62/SQSTM1 forms protein

(H) Identification of a GABARAP-interacting region on TRIM5a. Bars indicate peptide spots on the arrays, with black bars indicating the strongest binding (corresponding to the charcoal-framed dots in G) and gray bars indicating weaker binding (dashed-framed dots in G). Highlighted sequences indicate canonical LIR (LIR-1) and alternative LIR (LIR-2) involved in GST-GABARAP binding. (I) Mutational analysis of RhTRIM5a LIR motifs for effects on TRIM5a binding to GABARAP by GST pull-down assay. (J) High-content image analysis of the punctate distribution (versus diffuse cytoplasmic distribution) of GFP-tagged WT or double-LIR mutant (LIR-1*&2*) RhTRIM5a. (K) High-content analysis of the abundance of punctate p-ULK1 (Ser-317) in HeLa cells expressing GFP alone or GFP-RhTRIM5a (WT or LIR-1*&2*). (L) High-content analysis of the abundance of endogenous LC3B puncta in cells as in (K). (M) Transfected 293T cells transiently expressing GFP alone or GFP-RhTRIM5a (WT or LIR-1*&2*) were infected with VSVG-pseudotyped HIV-1 and induced for autophagy by starvation for 4 hr, and levels of intracellular p24 were determined by immunoblotting. Data represent means ± SE; n R 3 experiments; *p < 0.05, yp R 0.05 (t test or ANOVA). See also Figure S7.

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