Current Biology 24, 951–959, May 5, 2014 ª2014 Elsevier Ltd All rights reserved

http://dx.doi.org/10.1016/j.cub.2014.03.038

Article Regulation of Clathrin-Mediated Endocytosis by Dynamic Ubiquitination and Deubiquitination Jasper S. Weinberg1 and David G. Drubin1,* 1Department of Molecular and Cell Biology, 16 Barker Hall, University of California, Berkeley, Berkeley, CA 94720-3202, USA

Summary Background: Clathrin-mediated endocytosis in budding yeast requires the regulated recruitment and disassociation of more than 60 proteins at discrete plasma membrane punctae. Posttranslational modifications such as ubiquitination may play important regulatory roles in this highly processive and ordered process. However, although ubiquitination plays an important role in cargo selection, functions for ubiquitination of the endocytic machinery are not known. Results: We identified the deubiquitinase (DUB) Ubp7 as a late-arriving endocytic protein. Deletion of the DUBs Ubp2 and Ubp7 resulted in elongation of endocytic coat protein lifetimes at the plasma membrane and recruitment of endocytic proteins to internal membranes. These phenotypes could be replicated by expressing a permanently ubiquitinated version of Ede1, the yeast Eps15 homolog, which is implicated in endocytic site initiation, whereas EDE1 deletion partially suppressed the DUB deletion phenotype. Both DUBs are capable of deubiquitinating Ede1 in vitro. Conclusions: Deubiquitination regulates formation of endocytic sites and stability of the endocytic coat. This regulation appears to occur through Ede1, because permanently ubiquitinated Ede1 phenocopies deletion of UBP2 and UBP7. Moreover, incomplete suppression of the ubp2D ubp7D phenotype by ede1D indicates that ubiquitination and deubiquitination are likely to regulate additional components of the endocytic machinery. Introduction Clathrin-mediated endocytosis in yeast and mammals requires the coordinated actions of more than 60 different proteins at plasma membrane foci [1–3] (see Figure S5A available online shows a schematic of yeast endocytosis). Although the order of arrival and departure of these proteins has been described with high precision, the role of posttranslational modifications in regulating the assembly and disassembly of endocytic machinery proteins has been less fully explored. A role for the Ark1/Prk1 protein kinases has been established [4–8], yet how dynamic ubiquitination and deubiquitination might regulate the endocytic machinery is not known. A role for ubiquitin as a signal for internalization of cargo molecules is well established [9–12], as is a role for deubiquitinating enzymes in the sorting of internalized receptors [13, 14]. The role of arrestin-like proteins and other endocytic proteins as specific adaptors that promote ubiquitination and internalization of specific cargos has also been explored [15]. However, although there is some evidence that

*Correspondence: [email protected]

ubiquitination and ubiquitin-binding domains may help hold components of the endocytic machinery together [16], how ubiquitination affects the dynamics and function of the endocytic machinery has not been as well studied. A mutant of Rvs167 that lacks a ubiquitinated lysine was reported to have no endocytic phenotype [17], whereas ubiquitination of Eps15 (homologous to yeast Ede1) has been proposed to control an intramolecular interaction [18–20]. The ubiquitin-like domain of the E3 ligase parkin has been shown to bind to Eps15 in a ubiquitin-interacting-motif-dependent manner and to be required for Eps15 ubiquitination, a modification that delays cargo internalization [20]. Because the yeast genome encodes 43 E3 ubiquitin ligases and 19 deubiquitinases (DUBs) [21], assigning endocytic functions to any given enzyme can be complicated as a result of compensatory effects from other enzymes when one is mutated, as well as secondary effects due to participation of enzymes in multiple processes. The yeast Nedd4 homolog Rsp5, a homologous to the E6AP carboxyl terminus domain E3 ligase, is known to ubiquitinate endocytic proteins and endocytic cargos [16, 17, 22–24]. Rsp5’s C2 domain can bind to lipids and its WW domains bind to proteins, including adaptors such as the arrestin-like proteins [15, 25, 26]. Temperature-sensitive mutations in Rsp5 cause fluid-phase endocytosis defects [25]. Rsp5 reportedly has physical interactions with Rvs167, Las17, Lsb1, and Lsb2 and genetic interactions with arp2, end3, sla2, las17, and ede1 mutants. RSP5 mutants are sensitive to the actin drug latrunculin A and have actin organization defects [27, 28]. These data suggest that Rsp5 has a role in endocytosis beyond modifying cargo as a signal for internalization. Rsp5 performs a coupled monoubiquitination of Vps9 wherein the substrate binds to a ubiquitin modification on the ligase before being ubiquitinated [29], and Rsp5 may likewise ubiquitinate Ede1 after it binds to the modified ligase, as has been proposed for Nedd4 and Eps15 [19]. Rsp5 is an essential protein also involved in RNA export, regulation of fatty acid biosynthesis, mitochondrial organization, and DNA repair [30–33]. Rsp5 localization to the plasma membrane, to Abp1-red fluorescent protein (RFP) punctae, and to intracellular punctae has been reported for the plasmid-based overexpression of the full-length protein and for fragments containing the C2 and WW domains [27, 31, 34]. Localization to invaginations was observed by immunoelectron microscopy of plasmid-expressed hemagglutinin-tagged Rsp5 [34]. Cortical localization of the full-length protein expressed at approximately endogenous levels was observed in a temperature-sensitive SLA2 mutant that is severely defective in endocytosis [34]. These observations suggest that Rsp5 is located at the plasma membrane, at least transiently. Rsp5 can monoubiquitinate substrates and preferentially assembles K63 chains over K48 chains, an action that is antagonized by the physically associated DUB Ubp2 [35, 36]. Ubp2 specifically degrades K63 chains, which are known to be important for endocytosis [35–40], making Ubp2 a strong candidate for regulating nonproteasome-related ubiquitination of the endocytic machinery. The DUB Ubp7 was identified via phage display as an interaction partner for the SRC

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(A) Ubp7DUCH-GFP localizes to cortical punctae. (B) Dual-color microscopy reveals that Ubp7DUCH-GFP and GFP-Ubp7 colocalize with Ede1-RFP or Sla1-mCherry at endocytic sites and arrive late in the endocytic pathway after actin assembly begins. Ubp7ΔUCH (C) Kymographs of Ubp7DUCH-GFP indicate that Ubp7ΔUCH-GFP Ubp7 localizes to the plasma membrane for a Merge Ubp7ΔUCH Ede1- Merge -GFP Ubp7ΔUCH Sla1short time (w13 s) and does not internalize with -GFP mCherry RFP -GFP the budding vesicle. Original Adjusted D E (D) In an sla2D mutant, endocytosis is blocked Ubp7ΔUCH Ubp7ΔUCH-GFP Sac6-RFP Merge rvs167Δ -GFP and long actin tails are formed associated with endocytic proteins trapped at the plasma memsla2Δ brane as actin subunits continuously flux through sla1Δ the network. Actin-binding proteins such as Sac6 decorate the tail, whereas Ubp7DUCH-GFP is ark1Δ prk1Δ Wild-Type (galactose) Wild-Type Wild-Type F sla2Δ confined to the plasma membrane, similar to endocytic coat proteins. (E) Ubp7DUCH-GFP cortical localization is GFP-Rsp5 reduced in cells lacking SH3 domain proteins. Original and brightness/contrast-enhanced images are shown. (F) GFP-Rsp5 primarily localizes to internal punctae, but in sla2D and ark1D prk1D mutants, which have strong endocytic blocks, GFP-Rsp5 also appears on the plasma membrane. Similar to the endocytic mutants, in cells grown in galactose, GFP-Rsp5 localizes to both internal punctae and to the plasma membrane. Line scans of wild-type cells show an even fluorescence punctuated by internal punctae, whereas line scans of sla2D, ark1D prk1D, or cells grown in galactose show strong peaks at the edges of cells, consistent with a plasma membrane localization. Cortical GFP-Rsp5 localization can be observed in buds but not mother cells of some wild-type cells grown in glucose (far right frames, Eclipse Ti). Scale bars, 1 mm (A, D, and F) and 200 nm (C). See also Figure S1.

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homology 3 (SH3) domains of multiple endocytic proteins [41], and it was enriched with many well-characterized endocytic proteins when Las17 (yeast Wiskott-Aldrich syndrome protein [WASP])-coated beads from which actin tails were assembled in yeast extracts were isolated [42]. This study reports that the DUB Ubp7 localizes to endocytic sites late in the pathway and that both Ubp2 and Ubp7 are capable of deubiquitinating Ede1 in vitro. Although Ede1 is more highly ubiquitinated in ubp2D ubp7D cells and both ubp2D ubp7D phenotypes are replicated by expression of permanently ubiquitinated Ede1, deletion of EDE1 in ubp2D ubp7D cells does not suppress a cytoplasmic punctae phenotype, suggesting that other components of the endocytic machinery involved in site initiation and coat stabilization are also regulated by ubiquitination. Results Ubp7 Localizes to Endocytic Sites, and Rsp5 Can Be Trapped at the Plasma Membrane An N-terminal fusion of GFP to Ubp7, expressed at the UBP7 chromosomal locus from the endogenous promoter, localizes to dynamic plasma membrane punctae, as does a C-terminal fusion that truncates the ubiquitin C-terminal hydrolase (UCH) (catalytic) domain (Figures 1A–1C; Figure S1A). Because Ubp7 is expressed at very low levels [43], making detection challenging, and because the DUCH-GFP fusion is significantly brighter than the N-terminal fusion (Figure S1A), the latter fusion protein was often used in this study. The dynamics and internalization of Sla1-mCherry, an endocytic adaptor protein, are normal in strains expressing either GFP-tagged Ubp7 (Figure S1B). Ubp7DUCH-GFP punctae have a short lifetime of

w13 s and do not appear to internalize with the nascent vesicle, remaining at the plasma membrane for their entire lifetime (Figure 1C). This behavior is shared with proteins involved in actin polymerization, such as Bzz1, Las17, and Myo3/5, but is distinct from coat and adaptor proteins such as Sla1p that internalize with the vesicle [1]. Ubp7DUCH-GFP and GFP-Ubp7 punctae both colocalize with endocytic markers Sla1-mCherry, Ede1-RFP, and Abp1RFP (Figure 1B; Figure S1A). Abp1-RFP is generally detectable before the arrival of either GFP-labeled Ubp7. However, labeled Ubp7 is extremely dim due to very low protein levels [43], making it difficult to precisely determine which protein arrives first. In sla2D cells, which have a strong endocytic block and form long actin tails due a disruption of the link between the endocytic coat and actin filaments [44], Ubp7 localizes with the coat proteins at the plasma membrane, and not with the actin tails, where many actin-binding proteins localize (Figure 1D). In cells treated with the actin monomer-sequestering drug latrunculin A, Ubp7DUCH-GFP localizes to cortical punctae even after actin has depolymerized (Figure S1C), a behavior shared with coat and adaptor proteins. The prolinerich region of Ubp7 interacts with the SH3 domains of endocytic proteins [41]. Surprisingly, single deletion mutants of any of several of these SH3-containing endocytic proteins greatly reduce Ubp7DUCH-GFP cortical localization (Figure 1E; Figure S1D), suggesting that the SH3-containing endocytic proteins recruit Ubp7 to the plasma membrane. Neither N- nor C-terminal GFP-tagged versions of Ubp2 revealed any specific localization, consistent with results of large-scale localization studies [21]. C-terminal tags fused to Rsp5 are lethal [45, 46], but N-terminal fusions as the sole source of Rsp5 allow for normal growth

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[35] (Figure S1E). N-terminal GFP fusions localize diffusely to the cytoplasm and to small, fast-moving internal punctae and to larger slower-moving punctae (Movie S1). Using an IX81 microscope (Olympus), we did not observe plasma membrane localization under standard growth conditions for chromosomally integrated GFP-Rsp5 driven by either the glyceraldehyde-3-phosphate dehydrogenase or transcription elongation factor promoters (Figure 1F, far left panel). However, cortical localization was observed preferentially at the tips of medium- and small-budded cells using an Eclipse Ti microscope (Nikon) equipped with a more sensitive camera (Figure 1F, far right panels). Galactose promoter-driven, plasmid-borne, full-length Rsp5 or a protein fragment lacking the catalytic domain was previously reported to localize to both the plasma membrane and internal punctae [27, 31, 34]. When cells were grown overnight in galactose, constitutively expressed GFP-Rsp5 was strongly localized to the plasma membrane of mother cells and buds as well as to internal punctae (Figure 1F). However, although partial colocalization between GFP-Rsp5 and endocytic patch proteins was observed in these situations, GFP-Rsp5 localized stably to large portions of the plasma membrane, whereas endocytic patches are small, discrete, and dynamic. Any colocalization appears to be coincidental rather than reflecting recruitment of GFP-Rsp5 to endocytic patches. There was no detectable colocalization of dynamic GFP-Rsp5 punctae with Abp1-RFP patches by epifluorescence or Sac6-RFP patches by epifluorescence or total internal reflection fluorescence microscopy (TIRFM). We therefore hypothesized that Rsp5’s interactions with substrate endocytic proteins may be transient and thus difficult to visualize. Endocytic mutants sla2D and ark1D

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(A) Cortical lifetimes of many endocytic proteins, especially proteins in the coat module, are extended significantly (p % 0.05, t test) in ubp2D ubp7D double mutants. Only modest effects were observed for proteins in laterarriving modules (n > 100 patches). Error bars show 61 SD. (B) Example kymographs for Sla1-GFP in wildtype and double mutant cells. Mutant cells have longer patch lifetimes. Time bar is 20 s.

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prk1D cause strong endocytic blocks, so we imaged GFP-Rsp5 in these mutants to test whether it gets trapped at the cell cortex. We observed a strong cortical fluorescent signal in these mutants on both mother cells and buds (Figure 1F), suggesting that GFP-Rsp5 is trapped at the plasma membrane when endocytosis is blocked.

Deletion of DUB Genes Extends Endocytic Coat Lifetimes and Causes Recruitment of Endocytic Proteins to Early Endosomes The cortical lifetimes of GFP- or RFP-labeled endocytic proteins were measured in wild-type and ubp2D ubp7D cells (Figure 2A). Although the phenotypes described below were observed in both ubp2D and ubp7D single mutant cells (data not shown), the double mutants had stronger phenotypes than either single mutant. Lifetimes were extended for many endocytic proteins, especially those in the coat module, and to a lesser extent, for proteins in the WASP/Myo and scission modules, which arrive later (see Figure S5A). The fluorescently labeled coat proteins internalized normally (Figure 2B), even when their lifetimes were significantly extended (p % 0.05). In addition to extended cortical lifetimes, endocytic proteins appeared on ectopic cytoplasmic patches in ubp2D ubp7D cells (Figure 3A; Figure S2A). Under normal circumstances endocytic coat proteins appear at cortical patches and disappear upon uncoating after traveling a few hundred nanometers into the cell [1]. In ubp2D ubp7D cells, endocytic proteins localize to cytoplasmic punctae as well as to the plasma membrane. These punctae were stained by N-(3-triethylammoniumpropyl)-4-(p-diethylaminophenyl-hexatrienyl) pyridinium dibromide (FM4-64) within 3 min of addition of the lipid dye to the media, suggesting that the endocytic proteins assemble on early endosomes (Figure 3C). All of the labeled endocytic proteins tested, including early, coat, and actin module proteins, appeared on the internal patches (Figure 3A). Internal patches generally move rapidly within the cytoplasm and then disappear, either by uncoating or by moving out of the plane of focus. In cells expressing both Sla1-GFP and Abp1RFP, internal punctae are labeled by Sla1-GFP that then is joined by Abp1-RFP. Then, both proteins disappear (Figure 3B). These internal punctae may represent endocytic sites inappropriately initiated on early endosomes in response to an ubiquitin signal that has not been removed due to absence of Ubp2 and Ubp7.

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Figure 3. ubp2D ubp7D Double Mutants Have Internal Patches (A) Double mutant cells have internal patches to which multiple different endocytic proteins from several different modules get recruited in the normal sequence. These patches are dynamic and mobile, appearing and disappearing and moving around inside the cell. Arrows point to internal punctae labeled by various fluorescent endocytic proteins. Scale bar, 1 mm. (B) Dual-color microscopy of endocytic patches demonstrates that they behave similarly to cortical actin patches. Sla1p appears first, followed by Abp1, which marks actin, followed by the disappearance of the patch. Frames are 1 s apart. (C) Within 3 min of addition of FM4-64, colocalization is observed between FM4-64 punctae and cytoplasmic punctae marked by Sla2-GFP. Arrows point to internal punctae that are labeled with FM4-64 within 3 min of addition of the lipid dye and are labeled by the fluorescent endocytic coat protein Sla2-GFP. Scale bar, 1 mm. See also Figures S2 and S5.

Ubiquitination of Ede1 Is Increased in ubp2D ubp7D Cells The early endocytic protein Ede1 is a strong candidate for regulation by ubiquitination. Ede1 is known to be ubiquitinated by Rsp5, and it may bind intramolecularly to its own ubiquitin modification [16]. Additionally, Ede1 arrives early in the endocytic pathway, and it is important for endocytic site initiation [47], a process that may be perturbed in ubp2D ubp7D cells, as evidenced by the formation of ectopic cytoplasmic punctae. In order to test whether deletion of the two DUB genes changes the ubiquitination state of Ede1, we performed nickel affinity purifications of ubiquitin tagged with six histidines (His6-Ub) proteins from wild-type and DUB-deleted strains that had all endogenous ubiquitin genes deleted and expressed His6-myc-ubiquitin as the sole source of ubiquitin and then probed immunoblots for Ede1. More Ede1 was recovered from the ubp2D ubp7D strain than from the wild-type strain in four separate experiments, indicating that these DUBs control Ede1 ubiquitination levels (Figure 4A; Figures S3A and S3D). Both DUBs Are Capable of Deubiquitinating Immunoisolated Ede1 In order to directly test whether ubiquitinated Ede1 is a substrate for the DUBs, we performed in vitro deubiquitination experiments on immunoisolated Ede1. In order to increase the quantity of ubiquitinated Ede1, we used sla2D Ubp7DUCHGFP cells, which have a strong endocytic block due to a disruption of the link between actin and the clathrin coat [44]. Our data suggest that Ede1 becomes deubiquitinated late in endocytosis, and we reasoned that ubiquitinated Ede1 may accumulate in cells blocked at an intermediate stage of endocytosis. Ede1 isolated from sla2D cells runs as a ladder with at least six different bands. Anti-ubiquitin antibodies

only react with the upper bands, demonstrating that they represent ubiquitinated Ede1 (Figure 4B). Immunoisolated Ede1 was incubated with purified glutathione S-transferase (GST)-Ubp2 or GST-Ubp7 or with a combination of both of the active site C/S mutants. Either wild-type DUB was capable of deubiquitinating Ede1 (Figure 4C; Figure S3C), although GST-Ubp2 was considerably more active than GSTUbp7 as assayed by deubiquitination of Ede1 (Figure 4C) and Ub-AMC (Figure S3B, Ubp7 graph uses twice as much protein and Ub-AMC). Although both wild-type DUBs were capable of deubiquitinating Ede1, the inactive C/S mutants were not (Figure 4C; Figure S3C). Expression of an Ede1-Ub Fusion Protein Phenocopies a ubp2D ubp7D Double Mutant Similar to ubp2D ubp7D cells, expression of Ede1 carrying a C-terminal ubiquitin fusion, in the absence of endogenous Ede1, caused the appearance of cytoplasmic punctae marked by fluorescently tagged endocytic proteins, and extended the lifetimes of coat proteins at plasma membrane-associated endocytic sites (Figures 5A; Figures S2A, S2B, and S4). Although expression of the Ede1-Ub fusion was sufficient to cause both phenotypes, Ede1 was not necessary for the cytoplasmic punctae phenotype in ubp2D ubp7D cells because deletion of EDE1 did not suppress the appearance of internal punctae (Figures 5A; Figure S2A). However, deletion of EDE1 did suppress the extended coat protein lifetime phenotype (Figure S2B). This result is consistent with the observation that deletion of EDE1 reduces the cortical lifetime of coat and WASP/Myo proteins [47]. The coat protein lifetime increase seen in Ede1-Ub cells is not as strong as that seen in ubp2D ubp7D cells, further suggesting that Ede1 is not the only endocytic target of the DUBs. Nevertheless, expression of Pan1-Ub, another yeast protein with homology to mammalian

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Ede1 anti-ubiquitin (FL-76) Figure 4. Evidence that Ede1 Is Deubiquitinated by Ubp2 and Ubp7 (A) His6-ubiquitinated proteins were purified from UBP2 UBP7 (WT) and ubp2D ubp7D (DD) cells, and the proteins eluted from nickel columns were immunoblotted to detect Ede1. More Ede1 was purified in ubp2D ubp7D cells, suggesting that Ede1 is more highly ubiquitinated in mutant cells. This experiment was repeated four times with similar results. (B) Equal volumes of immunoisolated Ede1 were run on a 7% polyacrylamide gel and probed with either anti-Ede1 or an anti-ubiquitin (FL-76) antibody. Higher molecular weight bands correspond to ubiquitinated Ede1. (C) Immunoisolated Ede1 was incubated at room temperature with GST-Ubp2, GST-Ubp7, or both GST-Ubp2 C745S and GST-Ubp7 C618S, and then separated on a 7% gel and probed with an anti-ubiquitin antibody (FL-76). The amount of ubiquitinated Ede1 was greatly reduced upon incubation with GST-Ubp2; somewhat reduced following incubation with GST-Ubp7, which had less activity against Ub-AMC (Figure S3B); and not deubiquitinated by the catalytically dead C/S mutants. See also Figure S3.

Eps15 [48–50], did not replicate either phenotype (Figures S2A and S2C). To determine if internal patches are formed de novo or if they are formed through a failure in vesicle uncoating, 11– 12 3 0.35 mm z-slices were collected every second for 30 s in ubp2D ubp7D cells expressing Ede1-GFP or Sla1-GFP. Internal patches were tracked to determine their point of origin. For both Ede1-GFP and Sla1-GFP, internal patches appeared de novo rather than being associated with an endocytic internalization event at the plasma membrane (Figure S5B). These data support the conclusion that internal patches represent ectopic endocytic site initiation rather than an uncoating defect (Figure 5B). Discussion The data presented here implicate dynamic ubiquitination and deubiquitination of the endocytic machinery in regulation of endocytic coat assembly and disassembly. The recruitment of a DUB during the late stages of clathrin-mediated endocytosis implicates ubiquitin removal in a late process like endocytic coat disassembly, whereas the appearance of endocytic proteins on internal membranes in ubp2D ubp7D and Ede1-Ub cells suggests that ubiquitin might be a signal for coat assembly or site initiation (Figure 5B). It is intriguing that the recruitment of Ubp7DUCH-GFP to the plasma membrane can be reduced by deleting any of several

SH3 domain-containing endocytic proteins (Figure S1D) that arrive prior to Ubp7. Perhaps Ubp7 is recruited when other proteins with proline-rich regions, such as Gts1, Scd5, and Aim21, begin to leave the patch [41], or perhaps Ubp7 only associates with endocytic sites after a critical number of SH3 domain-containing proteins have been recruited. Once recruited, late in the endocytic pathway, Ubp7 presumably removes ubiquitin from endocytic proteins, an activity that it can perform on ubiquitinated Ede1 (Figure 4C). Increased lifetimes of coat proteins, but not actin, in endocytic ubiquitination and deubiquitination mutants suggest that the mechanisms regulating coat formation using ubiquitination and deubiquitination do not regulate endocytic actin polymerization or vesicle scission. The appearance on internal membranes of endocytic proteins that exhibit the normal ordered arrival and departure observed on the plasma membrane suggests that deleting the two DUBs causes endocytic sites to be assembled inappropriately on early endosomes and that these sites then proceed at least partially through the normal sequence of endocytic events. Our microscopy indicates that the internal punctae form de novo and are not a result of a failure to uncoat internalizing vesicles. We hypothesize that deletion of the DUBs leaves behind ubiquitin as a signal on some proteins, resulting in aberrant initiation of endocytosis on early endosomes. This signal could be ubiquitination of the endocytic machinery or transmembrane cargo molecules. However, the ability of the Ede1-Ub fusion protein

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Figure 5. Permanently Ubiquitinated Ede1 Produces Internal Patches (A) Expression of Ede1-Ub results in internal patches similar to those found in ubp2D ubp7D double mutant cells. Arrowheads indicate internal punctae labeled by the fluorescent endocytic coat protein Sla2-GFP. Scale bar, 1 mm. (B) Model for regulation of endocytic patch dynamics by ubiquitination/deubiquitination. Ede1 is ubiquitinated by Rsp5, which facilitates endocytic site initiation and/or coat protein recruitment, and is deubiquitinated late in the process, just before scission, by Ubp2 or Ubp7, which facilitates disassembly of the coat. In ubp2D ubp7D cells or Ede1-Ub cells, Ede1 retains the ubiquitin and inappropriately initiates sites and promotes recruitment of endocytic coat proteins on early endosomes. See also Figures S2, S4, and S5.

to cause both extended coat lifetimes and recruitment of endocytic proteins to internal membranes implicates Ede1 as a functional target of the DUBs for coat regulation (Figure 5B) rather than, or in addition to, transmembrane cargo molecules. This conclusion is consistent with previous reports that Ede1 is ubiquitinated by Rsp5 [16], with our detection of multiple Ede1 species under certain conditions, with our recovery of more Ede1 from ubp2D ubp7D cells than from wild-type cells during His6-Ub purifications, and with our demonstration that the DUBs are capable of deubiquitinating Ede1 in vitro. The specific mechanism for the regulatory effect of ubiquitination on coat protein dynamics is not known. A proposed cargo transition point is thought to regulate the progression from site initiation to later stages [51–55]. Ede1 may bind ubiquitinated cargos [56] and is thought to be negatively regulated by its ubiquitin-associated (UBA) domain [16]. We propose that under normal conditions, the UBA domain of Ede1 is capable of either directly binding to and sensing cargo or indirectly influencing the cargo checkpoint by stabilizing the network of endocytic proteins as previously proposed [16]. Ede1 may act similarly to its mammalian homolog Eps15 and bind to its own ubiquitin modification via its C-terminal UBA domain [16, 18], and due to the intramolecular interaction be unable to perform its cargo-sensing function, extending the lifetime of endocytic coat proteins, but not the lifetimes of proteins arriving after the proposed transition point (Figure 2A; see Figure 5B for model). Ede1 is also extensively phosphorylated [57–59], raising the possibility that these modifications may function in a coordinated manner.

Evidence that Rsp5 performs an endocytic function comes from endocytic defects observed in rsp5 mutants [25, 28]. Although definitive proof of an E3 ubiquitin ligase at endocytic sites remains elusive, the fact that Rsp5 accumulates at the plasma membrane when endocytosis is perturbed suggests that it normally spends time at the plasma membrane and is removed by endocytosis via an unknown mechanism. The observation that GFP-Rsp5 is enriched on the plasma membrane of buds is intriguing. Perhaps more Rsp5 is recruited because endocytosis occurs preferentially in buds. Although it had previously been shown that Rsp5 is responsible for the Ede1 ubiquitination [16], our data indicate that Ubp2 and Ubp7 are directly responsible for deubiquitination of Ede1 and likely other endocytic proteins. Moreover, the data presented here implicate dynamic ubiquitination and deubiquitination of the endocytic machinery, distinct from the previously documented ubiquitination and deubiquitination of cargo molecules, in regulation of endocytic coat assembly, maintenance, and disassembly [11–14, 24, 38, 60]. Because all of the implicated components are highly conserved, we expect that these results will apply broadly. Experimental Procedures Strains Yeast strains used in this study are listed in Table S1. Gene deletions were generated by replacing the gene open reading frame with the Candida glabrata LEU2 or URA3, hphNT1, or KanMX4 cassette. C-terminal GFP tags were integrated as previously described [61]. The N-terminal GFP tag

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for Rsp5 was integrated as previously described [62]. N-terminal tagging of Ubp7 was performed as previously described [63]. Microscopy Yeast strains for imaging were grown to log phase at 30 C (25 C for sla2D and ark1D prk1D) in synthetic media lacking tryptophan and immobilized on concanavalin A-coated coverslips. For FM4-64 experiments a flow cell was constructed with double-stick tape, allowing for addition of FM4-64 or DMSO while imaging. Microscopy was performed on IX71 and IX81 microscopes (Olympus) with 1003/numerical aperture (NA) 1.4 objectives and Orca cameras (Hamamatsu) at 25 C. Simultaneous two-color imaging was performed using an argon-ion laser (CVI Melles Griot) to excite GFP and a 561 nm diode-pumped solid-state laser (CVI Melles Griot) to excite RFP. TIRFM was performed using an IX81 microscope equipped with a 1003/NA 1.65 objective with independently adjustable 488 and 561 nm lasers. Images were acquired with a frame rate of 1 frame/s using MetaMorph software (Molecular Devices), patch lifetimes were calculated using ImageJ (NIH), and graphs and statistics were generated using Excel (Microsoft). GFP-Rsp5 imaging and high time resolution stacks were collected using an Eclipse Ti microscope equipped with a 1003 NA1.4 objective, TiND6-PFS Ti-E Perfect Focus Unit (Nikon), Neo sCMOS camera (Andor), PZ-2000 piezo stage (Applied Scientific Instrumentation), SPECTRA X light engine (Lumencor), and an incubator (InVivo Scientific) using MetaMorph software and were analyzed using ImageJ. His6-Ub Purification His6-Ub purifications were adapted from Ziv et al. [23]. Yeast were grown to log phase in 500 ml of YPD at 30 C and collected via centrifugation at 3,500 rpm in an SLA-3000 rotor (Sorvall). Cells were resuspended in a small volume of water and frozen in liquid nitrogen. Trichloroacetic acid (TCA) (Fisher Scientific) was added to 20%, and cells were lysed by bead beating in a vortex (4 3 30 s with 30 s on ice between). Supernatants were collected by centrifugation (1 min, 1,000 3 g), beads were washed with 12% TCA, and the wash was collected by centrifugation (1 min, 1,000 3 g). Supernatants were incubated on ice for 30 min, pellets were collected by centrifugation (15 min; 14,000 rpm; 4 C), and the pH was adjusted using NH4OH. Pellets were resuspended in urea buffer (8 M urea, 20 mM Tris [pH 8.0], 100 mM K2HPO4, 10 mM imidazole, 100 mM NaCl, and 0.1% Triton X-100) and incubated on ice for 30 min. Supernatants were collected by centrifugation and incubated with nickel-nitrilotriacetic acid beads (QIAGEN) overnight at 4 C. Beads were collected by centrifugation and washed two times with urea buffer; the flowthrough was reapplied to beads and rotated at room temperature for 3 hr. Beads were collected by centrifugation and washed twice with wash buffer 1 (20 mM Tris [pH 8.0], 100 mM K2HPO4, 20 mM imidazole, 100 mM NaCl, and 0.1% Triton X-100), twice with wash buffer 2 (20 mM Tris [pH 8.0], 100 mM K2HPO4, 20 mM imidazole, and 100 mM NaCl), eluted with low pH buffer (20 mM Tris [pH 4.5], 100 mM K2HPO4, 150 mM imidazole, and 100 mM NaCl), and then analyzed by SDS-PAGE. Rabbit anti-Ede1 and was a generous gift from Linda Hicke. Anti-rabbit horseradish peroxidase was purchased from GE Healthcare, and blots were read using a ChemiDoc XRS+ system (Bio-Rad). GST-DUB Purification UBP2 and UBP7 open reading frames were cloned into pGEX-2T (GE Healthcare) and expressed in BL21 cells as previously described [64]. After induction, the bacteria were frozen in liquid nitrogen; resuspended in HEN (50 mM HEPES [pH 7.5], 1 mM EDTA, and 200 mM NaCl) supplemented with 2 mM phenylmethanesulfonylfluoride, 4 mM NEM (Sigma), and 23 Protease Inhibitor Cocktail Set II (Calbiochem); and lysed by six 10 s pulses of sonication with 30 s on ice between pulses. Cellular debris was pelleted twice by centrifugation at 14,000 rpm in a microcentrifuge at 4 C for 15 min. After addition of Triton X-100 to 1%, supernatants were rocked with glutathione beads (GE Healthcare) overnight. Beads were washed twice with HEN + 1% Triton, three times with HEN, and eluted three times for 30 min with HEN + 50 mM Tris (pH 8.0) + 20 mM glutathione (Sigma) with a final overnight elution. Catalytically dead mutants were purified similarly after mutagenesis of the pGEX-2T plasmids using a QuikChange Lightning kit (Agilent Technologies). DUBs were assayed in 100 ml reactions with final buffer concentrations of 50 mM Tris (pH 7.5), 1 mM dithiothreitol, 100 mg/ml BSA (New England Biolabs), and 100 nM or 200 mM (for Ubp7) Ub-AMC (Enzo Life Sciences) using a Victor3 1420 multilabel counter (PerkinElmer).

Ede1 Immunoisolation Immunoisolations were performed as previously described [52] using 100 optical density 600s of cells and anti-Ede1 antibodies (a kind gift from Linda Hicke). Lysis buffer was supplemented with 10 mM NEM and Protease Inhibitor Cocktail Set IV at 1:200. During the final wash step, the beads were divided into equal volumes for the subsequent in vitro deubiquitination reactions. In Vitro Deubiquitination Reactions Reactions were prepared with 10 ml of protein G beads from an Ede1 immunoisolation; 10 ng of purified DUB; buffer to final concentrations of 50 mM Tris [pH 7.5], 1 mM dithiothreitol, and 100 mg/ml BSA (NEB); and then rotated at room temperature for 60 min. Reactions were stopped by addition of 10 ml of 8 M urea (final concentration 1.25 M) and 12.5 ml of 53 SDS sample buffer (final concentration 13). Samples were analyzed by SDS-PAGE and immunoblotting with anti-Ede1 (Linda Hicke) and anti-ubiquitin (Santa Cruz Biotechnology FL-76) sera. Supplemental Information Supplemental Information includes five figures, two tables, and one movie and can be found with this article online at http://dx.doi.org/10.1016/j. cub.2014.03.038. Acknowledgments We are grateful to Dr. Georjana Barnes and the members of the D.G.D./ Barnes laboratory for helpful discussions, especially comments on this manuscript from Yidi Sun and Christa Cortesio. We thank Dr. Linda Hicke for plasmids LHP561 and LPH2798 and anti-Rsp5 and anti-Ede1 antibodies and Dr. Daniel Finley for strains SUB280 and SUB592. This work was supported by research grant R01 GM50399 from the NIH to D.G.D. Received: December 17, 2012 Revised: February 11, 2014 Accepted: March 12, 2014 Published: April 17, 2014 References 1. Kaksonen, M., Toret, C.P., and Drubin, D.G. (2005). A modular design for the clathrin- and actin-mediated endocytosis machinery. Cell 123, 305–320. 2. Kaksonen, M., Sun, Y., and Drubin, D.G. (2003). A pathway for association of receptors, adaptors, and actin during endocytic internalization. Cell 115, 475–487. 3. Taylor, M.J., Perrais, D., and Merrifield, C.J. (2011). A high precision survey of the molecular dynamics of mammalian clathrin-mediated endocytosis. PLoS Biol. 9, e1000604. 4. Zeng, G., Yu, X., and Cai, M. (2001). Regulation of yeast actin cytoskeleton-regulatory complex Pan1p/Sla1p/End3p by serine/threonine kinase Prk1p. Mol. Biol. Cell 12, 3759–3772. 5. Toret, C.P., Lee, L., Sekiya-Kawasaki, M., and Drubin, D.G. (2008). Multiple pathways regulate endocytic coat disassembly in Saccharomyces cerevisiae for optimal downstream trafficking. Traffic 9, 848–859. 6. Sekiya-Kawasaki, M., Groen, A.C., Cope, M.J., Kaksonen, M., Watson, H.A., Zhang, C., Shokat, K.M., Wendland, B., McDonald, K.L., McCaffery, J.M., and Drubin, D.G. (2003). Dynamic phosphoregulation of the cortical actin cytoskeleton and endocytic machinery revealed by real-time chemical genetic analysis. J. Cell Biol. 162, 765–772. 7. Cope, M.J., Yang, S., Shang, C., and Drubin, D.G. (1999). Novel protein kinases Ark1p and Prk1p associate with and regulate the cortical actin cytoskeleton in budding yeast. J. Cell Biol. 144, 1203–1218. 8. Chi, R.J., Torres, O.T., Segarra, V.A., Lansley, T., Chang, J.S., Newpher, T.M., and Lemmon, S.K. (2012). Role of Scd5, a protein phosphatase-1 targeting protein, in phosphoregulation of Sla1 during endocytosis. J. Cell Sci. 125, 4728–4739. 9. Hicke, L., and Riezman, H. (1996). Ubiquitination of a yeast plasma membrane receptor signals its ligand-stimulated endocytosis. Cell 84, 277–287. 10. Egner, R., and Kuchler, K. (1996). The yeast multidrug transporter Pdr5 of the plasma membrane is ubiquitinated prior to endocytosis and degradation in the vacuole. FEBS Lett. 378, 177–181.

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