JOURNAL OF CELL SCIENCE ACCEPTED MANUSCRIPT

© 2014. Published by The Company of Biologists Ltd.

TRE17/USP6 regulates ubiquitination and trafficking of cargo proteins that enter cells by clathrin-independent endocytosis

Yuji Funakoshi*†, Margaret M. Chou‡, Yasunori Kanaho†, and

Journal of Cell Science

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Julie G. Donaldson*

*Cell Biology & Physiology Center NHLBI, NIH Bethesda, MD 20891 USA †Department of Physiological Chemistry, Faculty of Medicine and Graduate School of Comprehensive Human Sciences, University of Tsukuba 1-1-1 Ten-nodai, Tsukuba, Ibaraki 305-8575, Japan ‡Department of Pathology and Laboratory Medicine, Children's Hospital of Philadelphia, University of Pennsylvania School of Medicine 3615 Civic Center Boulevard, Philadelphia, PA19104 USA

Running Head: TRE17 deubiquitinates CIE cargo proteins Key Words: ubiquitin, cargo sorting, ubiquitin-specific protease, lysosome Corresponding author email: [email protected] Word Count: 7,081 (excluding references)

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JCS Advance Online Article. Posted on 1 September 2014

Abbreviations: CIE, clathrin-independent endocytosis; CME, clathrin-mediated endocytosis; MHCI, major histocompatibility complex class I protein; TfR, transferrin receptor; USP,


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ubiquitin-specific protease

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Abstract Plasma membrane proteins that enter cells by clathrin-independent endocytosis (CIE) are sorted either to lysosomes for degradation or recycled back to the plasma membrane.

Expression of

some MARCH E-3 ubiquitin ligases promote trafficking of CIE cargo proteins to lysosomes by ubiquitinating the proteins. Here, we show that co-expression of the ubiquitin-specific protease


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TRE17/USP6 counteracts the MARCH-dependent targeting of CIE cargo proteins, but not transferrin receptor, to lysosomes, leading to recovery of the stability and cell surface level of the proteins.

The uiquitination of CIE cargo proteins by MARCH8 was reversed by TRE17,

suggesting that TRE17 leads to deubiquitination of CIE cargo proteins. The effects of TRE17 were dependent on its deubiquitinating activity and expression of TRE17 alone led to a stabilization of surface MHC Class I (MHCI), a CIE cargo, suggesting that deubiquitination of endogenous CIE cargo proteins promotes their stability.

This study demonstrates that cycles of

ubiquitination and deubiquitination can determine whether CIE cargo proteins are degraded or recycled.

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Introduction Protein levels are carefully monitored in cells through the regulated balance of protein synthesis and degradation. In general, soluble and membrane proteins are tagged for destruction by ubiquitination, which leads to degradation by the proteasome and lysosome, respectively. For cell surface proteins, ubiquitination can lead to post-endocytic sorting into intraluminal vesicles


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that form the multi-vesicular body (MVB), that later fuses with the lysosome (Piper and Lehner, 2011; Clague et al., 2012a). Sorting into the MVB requires ubiquitination of the cargo protein, and Endosomal Sorting Complex Required for Transport (ESCRT) complexes.

Ubiquitination

involves the covalent addition of ubiquitin typically onto lysine residues and this process involves the activity of E3 ligases. There are over 600 human E3 ligases, which are thought to exhibit specificity towards their substrates (Li et al., 2008). Conversely, deubiquitinating enzymes (DUBs) catalyze removal of ubiquitin. There are approximately 90 DUBs, but the extent to which DUBs have differential specificity is not clear (Clague et al., 2012b). Prior to degradation, the ubiquitin moiety on ubiquitinated proteins is removed by DUBs; this process allows for the recycling of the ubiquitin protein so it can be used for the next target. Recent evidence has emerged that in addition to providing a pathway for ubiquitin salvage, other DUBs can counteract or undo a ubiquitinated substrate thus rescuing it from degradation. We have been studying the trafficking and turnover of plasma membrane (PM) proteins that enter cells by clathrin-independent endocytosis (CIE). This mode of endocytosis occurs independently of clathrin and adaptor proteins and is used by a wide range of PM proteins (Naslavsky et al., 2004; Eyster et al., 2009).

Some CIE cargo proteins, such as major

histocompatibility complex Class I (MHCI), can be either recycled back to the PM or routed to late endosomes and lysosomes for degradation (Naslavsky et al., 2004). Other CIE cargo proteins, such as CD98 and CD44, are mostly recycled back to the PM with little routing to 4

degradation (Eyster et al., 2009; 2011). Members of the MARCH family of E3 ligases ubiquitinate CIE cargo proteins, causing them to be routed directly to late endosomes and lysosomes (Eyster et al., 2011).

This change in trafficking induced by expression of MARCH8

is observed for MHCI, but is most dramatic for CD98 and CD44, which normally avoid degradative compartments (Eyster et al., 2011).


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TRE17, also known as USP6, is a member of the family of ubiquitin-specific proteases (USP), the most abundant group of DUBs (> 50 members). TRE17 was originally identified as an oncogene (Nakamura et al., 1992). Later studies revealed that translocation of the TRE17 locus leads to overexpression of the wild type protein and is associated with two neoplasms, aneurysmal bone cyst (Oliveira et al., 2004a, 2004b, 2005; Panagopoulos et al., 2008) and nodular fasciitis (Erickson-Johnson et al., 2011). The USP domain of TRE17 is required for tumorigenesis (Ye et al., 2010; Pringle et al., 2012). However, relevant substrates have not been identified to date. TRE17 has another characteristic domain, the TBC (Tre-2/Bub2/Cdc16) domain, through which it binds to Arf6, a G protein associated with the CIE endosomal membrane system (Martinu et al., 2004). TRE17 co-localizes with Arf6 and CIE cargo proteins. TRE17 associates with GDP-bound Arf6 and promotes activation of Arf6 in vivo in a manner requiring its TBC domain (Martinu et al., 2004; Lau et al., 2010), and has been proposed to promote recycling of CIE cargo proteins. However, the role of the USP domain in TRE17's trafficking function has not been explored. In the current study, we re-examine the role of TRE17 in influencing CIE cargo protein trafficking. In particular, we investigate whether TRE17, through its USP activity, can counter the increased degradation of CIE cargo proteins triggered by MARCH expression.

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Results

TRE17 counteracts MARCH-dependent targeting of CIE cargo to late endosomes in a DUB activity-dependent manner. In our previous work, we demonstrated that trafficking of CIE cargo proteins is altered by


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expression of MARCH proteins through ubiquitination. We hypothesized that expression of TRE17 might affect ubiquitination-dependent CIE cargo protein trafficking through its DUB activity. To examine the effect of TRE17 on trafficking of CIE cargo proteins, we co-expressed TRE17 with the MARCH8 ubiquitin ligase in HeLa cells and followed the fate of internalized MHCI, a CIE cargo protein that is targeted by MARCH8 (Eyster et al., 2011). To track MHCI endocytosis and its intracellular trafficking, HeLa cells were incubated with monoclonal antibodies directed to the extracellular portion of the protein for 1 h to allow antibody-bound MHCI to enter the cells. Then, HeLa cells were treated with the proton ionophore NH4Cl for 2 h to neutralize the pH of the late endosome and block degradation in order to visualize cargo delivery to late endosomes.

As we reported previously, over-expression of MARCH8 caused

down-regulation of MHCI from the cell surface, with concomitant accumulation of the proteins in an enlarged juxtanuclear compartment (Fig. 1A, top panels). This compartment was co-stained with the late endosome/lysosome marker Lamp1 (Eyster et al., 2011) (data not shown), suggesting that MARCH8 targets MHCI to late endosomes for degradation. In clear contrast, most of cells co-expressing GFP-TRE17 and MARCH8 did not exhibit juxtanuclear accumulation of MHCI and instead maintained MHCI at the cell surface (Fig. 1A, middle, outlined with dashed lines), suggesting that TRE17 can suppress the function of MARCH8. In contrast, expression of a TRE17 point mutant that lacks DUB activity (TRE17/USP-) (Shen et al., 2005) failed to suppress the effect of MARCH8. Cells co-expressing TRE17/USP- and 6

MARCH8 were indistinguishable from those expressing MARCH8 alone (Fig. 1A, bottom). Quantification revealed that more than 90% of cells co-expressing MARCH8 with GFP or GFP-TRE17/USP- exhibited reduced surface labeling and increased juxtanuclear accumulation of MHCI (Fig. 1B). In contrast, only 15% of cells co-expressing MARCH8 and GFP-TRE17 exhibited reduced surface labeling and increased juxtanuclear accumulation of MHCI, as surface


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MHCI was once again apparent. These results suggest that TRE17 can counteract the effect of MARCH8 in a DUB-dependent manner. We previously identified a new group of CIE cargo proteins (CD44, CD98, and CD147) that follow a different intracellular itinerary from MHCI (Eyster et al., 2009, 2011). These cargoes largely avoid transport to degradative compartments and are instead recycled directly to the plasma membrane after internalization. We demonstrated that CD98 and CD44 are diverted to late endosome upon expression of MARCH8 (Eyster et al., 2011).

Therefore, we predicted

that TRE17 might also rescue them from a degradative fate. Similar to MHCI, CD98 delivery to late endosomes was increased and its surface level was decreased by the expression of MARCH8 (Fig. 1C, top, and Fig. 1D). This effect was suppressed by co-expression of TRE17 but not by TRE17/USP- (Fig. 1C, middle and bottom, and Fig. 1D). Similar results were obtained for CD44 (Fig. S1). The down-regulation of MHCI and CD98 and increased delivery to late endosomes by MARCH8 expression were also observed in a human bronchial epithelial cell line, Beas2B, and these effects were suppressed by expression of TRE17 (Fig. S2). Thus, TRE17 can alter the trafficking of two different types of CIE cargo proteins in two cell types that are targeted to late endosomes by MARCH8. We next examined whether TRE17 could reverse the effects of other MARCH family members on trafficking of CIE cargo proteins. Interestingly, different CIE cargoes exhibit distinct sensitivities to MARCH members: MHCI is down-regulated by MARCH1, MARCH4 7

and MARCH8, CD98 is down-regulated by MARCH1 and MARCH8, and CD44 is targeted only by MARCH8 (Eyster et al., 2011). Therefore, we tested whether TRE17 can also suppress the effects of MARCH1 and MARCH4. MARCH1 promoted targeting of CD98 to late endosomes, coincident with its diminished surface expression.

This was suppressed by the co-expression of

TRE17 but not by the TRE17/USP- mutant (Fig. S3A). Similarly, TRE17 reversed late


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endosomal targeting of MHCI induced by both MARCH1 (data not shown) and MARCH4 (Fig. S3B). Thus, TRE17 is capable of counteracting the function of multiple MARCH proteins that function in the CIE pathway.

Unique features of TRE17 determine its effects on CIE cargo trafficking We next examined whether other USPs could suppress the effect of MARCH on CIE trafficking, or whether this was a specific function for TRE17. USP8 is an endosomal DUB that has been shown to deubiquitinate and regulate the trafficking of several plasma membrane proteins, including EGF receptor, chemokine receptor CXCR4, Ca2+-activated K+ channel, and epithelial Na+ channel ENaC (Berlin et al., 2010a, 2010b; Balut et al., 2011; Zhou et al., 2013). Since USP8 is suggested to function at the plasma membrane and also at endosomal compartments, it is possible that USP8 might also regulate CIE cargo trafficking as TRE17 does. However, in contrast to TRE17, co-expression of USP8 did not suppress MARCH8-induced surface down-regulation and late endosomal accumulation of MHCI (Fig. 2A, middle) or CD98 (Fig. 2C, middle). As an additional specificity control, we also tested USP32. The TRE17 gene is derived from the segmental duplication of USP32 and TBC1D3, followed by their fusion (Paulding et al., 2003). Although USP32 possesses a distinct amino-terminus and lacks the TBC domain, its USP domain is 98% identical to that of TRE17. Strikingly, USP32 failed to rescue late 8

endosomal targeting of MHCI and CD98 induced by MARCH8 (Fig. 2A, C). Quantification demonstrates that more than 90% of cells co-expressing USP8 or USP32 with MARCH8 still exhibit surface down-regulation and late endosomal accumulation of both MHCI and CD98 (Fig. 2B, D). These results indicate that TRE17's ability to counteract the effects of MARCH proteins on CIE cargo trafficking is highly specific, and not merely a consequence of


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overexpression.

Furthermore, our data indicate that the N-terminal region of TRE17 containing

the TBC domain confers specificity to TRE17 in regulating trafficking of cargo through this pathway.

TRE17 does not affect trafficking of TfR In addition to CIE cargo proteins, we (Eyster et al., 2011) and others (Bartee et al., 2004; Nakamura et al., 2005; Fujita et al., 2013) previously showed that the transferrin receptor (TfR), a plasma membrane protein that enters cells by clathrin-mediated endocytosis (CME), is down-regulated by expression of some MARCH proteins.

Therefore, we examined whether

TRE17 would rescue the down-regulation of TfR mediated by expression of MARCH. When we examined the steady state level of TfR, we observed a significant down-regulation of TfR in MARCH8-expressing cells (Fig. 3A, middle row) as reported previously (Eyster et al., 2011; Fujita et al., 2013). In contrast to CIE cargo proteins, however, TRE17 failed to rescue this phenotype, with ~95% of cells co-expressing MARCH8 and TRE17 exhibiting down-regulation of TfR

(Fig. 3A, B). These results suggest that TRE17 cannot counteract the effect of

MARCH8 on TfR degradation and indicate that TRE17 has substrate specificity: TRE17 can rescue CIE cargoes that are targeted by MARCH but not TfR, a CME cargo.

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Compartmentalization of DUBs contributes to recognition of CIE cargo proteins To further investigate the substrate specificity of TRE17, we analyzed the subcellular localization of TRE17 and cargo proteins, MHCI, CD98 and TfR, by immunofluorescence staining. As reported previously (Masuda-Robens et al., 2003; Martinu et al., 2004), TRE17 localized at the plasma membrane and tubular endosomes (Figure 4). TRE17 exhibited


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significant co-localization with CIE cargo proteins, MHCI and CD98, at both of these locations. However, we did not observe co-localization of TRE17 with TfR (Figure 4). These observations raise the possibility that the specificity of TRE17 on CIE cargo trafficking is achieved through its co-compartmentalization with CIE cargo.

To test this, we

re-directed USP8, which failed to alter trafficking of CIE cargo (Fig. 2), to the CIE pathway by appending the C-terminal 20 amino acids of H-Ras (denoted, USP8-Ras C20).

This region of

H-Ras contains a farnesylation signal and mediates localization of H-Ras to the plasma membrane and clathrin-independent tubular endosomes and vesicles (Hancock et al., 1991; Porat-Shliom, et al, 2008; McKay et al., 2011). Furthermore, this peptide is sufficient to drive localization of GFP to the plasma membrane and clathrin-independent-derived endosomes and tubules. When expressed in HeLa cells, WT USP8 localized at cytosolic punctate structures and did not co-localize with CIE cargoes, MHCI and CD98 (Fig. 5A). In contrast, the USP8-Ras C20 chimera highly co-localized with both MHCI and CD98 at the plasma membrane and tubular endosomes, but not with TfR, a CME cargo (Fig. 5B), confirming that USP8-Ras C20 is targeted to the CIE endocytic pathway. Next, we examined whether USP8-Ras C20 alters the trafficking of CIE cargo proteins. In contrast to USP8, which did not suppress MARCH8-dependent down-regulation and accumulation of MHCI, co-expression of USP8-Ras C20 markedly suppressed the effect of MARCH8: nearly 70% of cells co-expressing USP8-Ras C20 recovered MHCI at the cell surface 10

(Fig. 6A, B). Similar results were obtained for CD98 (nearly 60% were recovered) (Fig. 6C, D). On the other hand, USP8-Ras C20 could not counteract the down-regulation of TfR caused by expression of MARCH8 (Fig. 6E, F), indicating that USP8-Ras C20 has similar substrate specificity as TRE17. Therefore, localization of DUBs, TRE17, USP8 and USP8-Ras C20, clearly correlated with the ability to alter the itinerary of cargo proteins. These results suggest


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that the substrate specificity against CIE cargo proteins is determined in part by sub-cellular localization of the DUB.

CIE cargo proteins are deubiquitinated in TRE17-expressing cells To test whether the function of TRE17 is mediated through deubiquitination of target cargo proteins, we examined whether ubiquitination of CIE cargo proteins by MARCH is reversed by TRE17. To facilitate detection of ubiquitination of CIE cargoes, we used C-terminally SNAP-tagged CD98. The SNAP tag is an engineered form of a 20 kDa DNA repair enzyme (O6-alkylguanine-DNA alkyltransferase) that binds covalently to O6-benzylguanine (BG) (Gautier et al., 2008), which can be attached to Alexa dyes and biotin. We first sought to confirm that trafficking of CD98-SNAP recapitulates that of the endogenous protein. HeLa cells expressing CD98-SNAP were exposed to BG-Alexa 594 for 1 h at 37 ºC and chased for 2 h in media containing 25 mM NH4Cl to follow the trafficking of CD98-SNAP. Similarly to endogenous CD98, CD98-SNAP was lost from the cell surface and accumulated at late endosomes upon MARCH8 overexpression (Fig. S4A, second row). This effect was counteracted by TRE17, but not TRE17/USP- (Fig. S4A, third and bottom rows). Thus, both MARCH8 and TRE17 regulate the trafficking of CD98-SNAP similarly to endogenous CD98.

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Next, we investigated the ubiquitination status of CD98-SNAP in cells expressing MARCH8 and TRE17. We previously demonstrated that MARCH8 induces the ubiquitination and degradation of both endogenous CD98 and CD98-SNAP (Eyster et al., 2011). To test whether TRE17 can reverse this, HeLa cells expressing CD98-SNAP were labeled with BG-biotin for 4 h, and biotin-labeled CD98-SNAP was isolated on streptavidin beads and


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detected by anti-ubiquitin blotting. To ensure that anti-ubiquitin signals are derived from CD98-SNAP and not co-purifying proteins, cell lysates were denatured by SDS and boiled before the pull-down. CD98-SNAP exhibited a low basal level of ubiquitination that was strongly enhanced by co-expressing MARCH8 (Fig. 7A, top panel, compare lanes 1 and 2). The smeared bands observed in the anti-ubiquitin blot were not detected in control cells (no CD98-SNAP) confirming that the band corresponds to ubiquitinated CD98-SNAP (data not shown). Concomitant with this increased ubiquitination, total levels of CD98-SNAP were dramatically reduced upon co-expression with MARCH8, consistent with its targeting for lysosomal degradation (Fig. 7A, second and third panels). When TRE17 was co-expressed with MARCH8, the smeared band observed in cells expressing MARCH8 alone was remarkably decreased (Fig. 7A, top panel, compare lanes 2 and 3).

In contrast, expression of the

TRE17/USP- mutant did not reduce MARCH8-dependent ubiquitination of CD98-SNAP (Fig. 7A, top panel, lane 4). Correlated with the level of ubiquitination, co-expression of TRE17 but not the USP- mutant partially restored levels of CD98-SNAP protein (compare lanes 1-4 in anti-SNAP blot). These results suggest that TRE17 counteracts the function of MARCH8 by promoting deubiquitination of CD98-SNAP. Furthermore, expression of other DUBs, USP8 and USP32, did not affect MARCH8-dependent ubiquitination significantly (lanes 5 and 6), suggesting that TRE17's effects on CD98-SNAP ubiquitination and protein levels are specific. Quantification of these results further supports the above observation. We measured intensities 12

of anti-ubiquitin blot and normalized to the amount of isolated CD98-SNAP to calculate the level of ubiquitination of CD98-SNAP (Fig. 7B). Expression of MARCH8 increased ubiquitination of CD98-SNAP more than 35-fold compared to the control (columns 1 and 2). Co-expression of TRE17 markedly decreased MARCH8-dependent ubiquitination (columns 2 and 3). On the other hand, the amount of ubiquitination of CD98-SNAP in cells co-expressing the TRE17/USP-,


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USP8 or USP32 was not significantly differed from that of cells expressing MARCH8 alone (columns 2, 4, 5 and 6). To determine whether TRE17's effects on ubiquitination and degradation could be extrapolated to other CIE cargo, we performed similar analysis of Tac, the alpha subunit of the interleukin 2 receptor. Tac is a CIE cargo protein that follows a similar intracellular trafficking itinerary as MHCI (Naslavsky et al., 2003). We appended the SNAP-tag to the amino-terminus of Tac and examined the trafficking and ubiquitination of SNAP-Tac.

Similar to the

observation for CD98-SNAP, SNAP-Tac was routed to late endosomes and the cell surface level was decreased by the expression of MARCH8 (Fig. S4B). These phenotypes were suppressed by the co-expression of TRE17 but not by the USP- mutant. When we examined ubiquitination of SNAP-Tac, two discrete bands and a faint smeared band that ran slightly above the size of SNAP-Tac were observed in cells expressing MARCH8 in anti-ubiquitin blots (Fig. 7C, top panel, compare lanes 1 and 2).

These bands were not observed in control cells, confirming that

the bands are ubiquitinated SNAP-Tac (data not shown). When TRE17 was co-expressed with MARCH8, ubiquitination of SNAP-Tac was strongly suppressed (compare lanes 2 and 3). However, expression of TRE17/USP-, USP8 and USP32 did not affect the ubiquitination of SNAP-Tac by MARCH8. Coupled to the ubiquitination status, the expression level of SNAP-Tac was markedly decreased in cells expressing MARCH8 alone and recovered in cells specifically upon co-expression of TRE17. Quantification of ubiquitinated SNAP-Tac gave 13

similar results as CD98-SNAP, with TRE17 but not TRE17/USP-, USP8 and USP32 decreasing MARCH8-dependent ubiquitination of SNAP-Tac (Fig. 7D). Taken together, these findings demonstrate that TRE17 promotes the deubiquitination of two different classes of CIE cargo proteins that are targeted by MARCH, leading to their enhanced stability through inhibition of

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MHCI is stabilized in TRE17-expressing cells


delivery to late endosomes.

MHCI in cells expressing TRE17 in order to examine its effect on the stability of CIE cargo.

Having determined that TRE17 can counteract the effect of MARCH that targets cargo proteins for degradation, we next examined whether expression of TRE17 alone could alter the stability of endogenous cargo proteins. We previously demonstrated that pulse-labeled MHCI proteins are routed to degradative compartments resulting in almost complete disappearance of the labeled proteins from the cell surface after 24 h. We followed the long-term fate of surface

Cells expressing TRE17 were incubated with anti-MHCI antibody on ice for 1 h to label the surface MHCI. Antibody was washed out and cells were either fixed immediately (0 h) or further incubated for 20 h at 37ºC. After incubation, cells were fixed and antibody remaining at the cell surface was detected by using secondary antibodies without permeabilizing the cells (Fig. 8A).

By measuring intensities of the cell surface MHCI as described in Materials and Methods,

we compared the surface level of MHCI among cells expressing GFP control, TRE17 wt and TRE17/USP- (Fig. 8B). At 0 h time point, the surface level of MHCI was slightly, but significantly, increased in cells expressing TRE17 but not in cells expressing TRE17/USP-. This indicates that TRE17 modestly up-regulates steady state levels of MHCI in a DUB-dependent manner. After 20 h of incubation, the cell surface MHCI labeled with the antibodies was dim in control (GFP) cells (Fig. 8A, right panels). However, in cells expressing 14

TRE17 wt, signal intensities of surface MHCI were higher than that of non-expressing cells.

In

contrast, cells expressing TRE17/USP- did not show significant differences in the amount of surface MHCI compared to non-expressing, control cells. Quantification of cell surface MHCI levels demonstrated that cells expressing TRE17 wt but not the USP- mutant have approximately 60% more surface MHCI compared to GFP control cells (Fig. 8B right). These results indicate

of the protein at the cell surface.

Taken together, our results suggest that TRE17 not only

counteracts the effects of over-expressed MARCH E-3 ligases, but also regulates the trafficking of endogenous CIE cargo proteins by promoting deubiquitination and thus blocking their degradation.


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that expression of TRE17 by itself can stabilize endogenous MHCI resulting in increased levels

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Discussion In this study we show that TRE17 counteracts the MARCH-dependent ubiquitination and down-regulation of CIE cargo proteins in a DUB activity-dependent manner.

Among several

USPs localized to the periphery, TRE17 was specifically able to regulate the trafficking of CIE cargo proteins upon overexpression. Together with our previous studies, our work indicates that


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trafficking of CIE cargoes can be regulated by reversible ubiquitination mediated by the coordinated activities of TRE17 and MARCH proteins. We showed previously that MARCH isoforms exhibit substrate specificity for the down-regulation of CIE cargo proteins: CD44 is down-regulated by over-expression of MARCH8, CD98 by MARCH1 and MARCH8, and MHCI by MARCH4 and MARCH8 (Eyster et al., 2011).

In contrast, TRE17 suppressed the effects of all these MARCH proteins, and

promoted recovery of CD44, CD98 and MHCI at the cell surface. Therefore, it is likely that TRE17 can widely recognize CIE cargo proteins that are ubiquitinated. On the other hand, TRE17 could not counteract the MARCH-dependent down-regulation of TfR, a CME cargo. Thus, TRE17 appears to selectively regulate CIE but not CME pathway cargoes. In support of this view, TRE17 highly co-localized with CIE cargo proteins, MHCI and CD98, but not with TfR, at the plasma membrane and tubular recycling endosomes that are characteristic structures for CIE trafficking pathways (Naslavsky et al.,2004; Martinu et al., 2004) (Fig. 4). Furthermore, we did not observe co-localization of TRE17 with AP-2, an adaptor protein subunit for CME (unpublished observations). The localization of TRE17 appears to be a key determinant of how it recognizes its substrates. The results of the USP8-Ras C20 chimera (Figs 5, 6) further support this possibility. Although USP8 itself does not affect CIE cargo trafficking, when it is targeted to CIE trafficking pathways by connecting H-Ras C-terminal farnesylation signal, the chimeric protein co-localized 16

with CIE cargoes and altered their itinerary.

These results suggest that a DUB relocalized to

CIE trafficking pathways obtains substrate specificity against CIE cargoes.

It is likely that

recruitment of the DUB to the CIE cargo-specific compartment is important to achieve its substrate specificity against CIE cargoes.

TRE17 is recruited to the plasma membrane from

which CIE cargo enter, and to endosomal compartments that are related to CIE trafficking


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pathways. Some factors of the CIE trafficking machinery might recruit TRE17 to the sites where cargo proteins are deubiquitinated. Interestingly, TRE17 is known to bind Arf6, a key regulatory factor for the CIE trafficking pathway. It has been shown that Arf6 and TRE17 co-localize at the plasma membrane and tubular recycling endosomes (Masuda-Robens et al., 2003; Martinu et al., 2004).

In addition, Rueckert and Haucke (2012) have shown that

subcellular localization of TRE17 depends on Arf6. Thus, Arf6 might serve to recruit TRE17 to target CIE cargo proteins for deubiquitination. We found that expression of TRE17 by itself can stabilize MHCI and increase the surface level of the protein. Although it has not been clarified whether endogenous MARCH proteins contribute to the turnover of MHCI proteins in HeLa cells, this result indicates that the trafficking of MHCI is regulated by endogenous ubiquitination to some extent. Expression of TRE17 likely deubiquitinates endogenously ubiquitinated MHCI and leads to its stabilization. Thus, the effect of TRE17 is not limited to suppression of over-expressed MARCH. Whether endogenous TRE17 contributes to stabilization of CIE cargo proteins remains to be determined. While TRE17 expression in normal tissues appears to be limited to testis (Paulding et al.,2003), low levels have been reported in HeLa cells.

We examined whether knock down

of TRE17 could affect trafficking of CIE cargo proteins in HeLa cells, but unfortunately we could not consistently detect substantial levels of endogenous TRE17 (unpublished observations). Nevertheless, we believe that the effects of TRE17 on CIE cargo trafficking are very likely 17

physiologically relevant since, even upon overexpression, other USPs were incapable of regulating CIE cargo as TRE17 does. Most notably, USP32, which shares 98% identity with TRE17 in the catalytic domain, failed to reverse the juxtanuclear accumulation of CIE cargo. Furthermore, the overexpression phenotype of TRE17 is highly relevant to oncogenesis, as TRE17 is translocated and vastly overexpressed in two human tumors.


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TRE17 was originally identified as an oncogene from human Ewing’s sarcoma (Nakamura et al., 1992) and is genetically linked to aneurysmal bone cyst (ABC) and nodular fasciitis (NF), tumors in which TRE17 is over-expressed by chromosomal translocation (Oliveira et al., 2004a, 2004b, 2005; Panagopoulos et al., 2008; Erickson-Johnson etal., 2011).

In addition, it has been

shown that injection of cells over-expressing TRE17 into nude mice induces formation of ABC-like tumors in a DUB activity-dependent manner (Ye et al., 2010). These lines of evidence strongly suggest that the DUB domain of TRE17 is required for tumorigenesis. Over-expression of TRE17 in various cells induces expression of MMP-9 and MMP-10, matrix metalloproteinases enriched in ABC tumors (Kumta et al., 2003). Induction of MMP-9 was mediated by NF-B, and inhibition of NF-B in TRE17-overexpressing cells dramatically reduced tumorigenesis (Ye et al., 2010; Pringle et al., 2012). Ruekert and Haucke (2012) have also shown that TRE17 is involved in the regulation of cell migration and cytokinesis through the Arf6-dependent pathway, indicating that TRE17 contributes to oncogenesis through the activation of Arf6. In addition to these previous studies, our findings may provide another mechanism by which over-expression of TRE17 contributes to tumorigenesis.

The expression

levels of CD44, CD98 and CD147, cell surface proteins that we previously identified as CIE cargo (Eyster et al., 2009), are known to be elevated in various types of cancers. CD44 is a hyaluronan receptor that mediates cell-matrix interactions and CD98 influences integrin trafficking and its signaling, both of which are related to growth, survival, motility, and invasion 18

of cancer cells (Zoller, 2011; Louderbough and Schroeder, 2011; Cantor and Ginsberg, 2012). CD147 also associates with integrins (Berditchevski et al., 1997) and elevated levels of cell surface CD147 on cancer cells induces production of MMPs in neighboring fibroblasts (Iacono et al., 2007).

By preventing the lysosomal degradation of these CIE cargo proteins, TRE17

would increase the cell surface level of these proteins, promoting tumor formation and


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progression. An examination of whether levels of these CIE cargo proteins are elevated in TRE17-expressing tumors and investigation of how they are up-regulated in the tumors will reveal pathogenesis of TRE17-dependent tumors and also help to elucidate physiological relevance of TRE17. To our knowledge, TRE17 is the first identified DUB that regulates trafficking of multiple CIE cargoes, including MHCI, CD98, CD44, and Tac. CIE is in general a non-selective process, however, once internalized, CIE cargo proteins follow different itineraries in the cell (Eyster et al., 2011). We recently identified a mechanism that facilitates recycling of specific CIE cargo proteins (Maldonado-Baez et al.,2013).

Reversible ubiquitination mediated by

MARCH and TRE17 might play an additional role in regulating trafficking of CIE cargo proteins, providing a quality control check at the level of the endosome. Elucidating how their activities are regulated will contribute to our understanding of regulatory mechanisms of CIE pathways.

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Materials and Methods

Antibodies and plasmids Mouse monoclonal antibodies to MHCI (clone W6/32; IgG2a), CD44 (clone BJ18; IgG1), and CD98 (clone MEM-108; IgG1) were purchased from Biolegend (San Diego, CA) and used for


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antibody internalization. Mouse monoclonal antibodies to transferrin receptor (clone 236-15375; igG1) were purchased from Invitrogen Molecular Probes (Grand Island, NY). Mouse monoclonal antibodies to GFP (clone B34; IgG1) and Chicken anti-GFP antibodies were purchased from Covance Research Products (Princeton, NJ) and Merk Millipore (Billerica, MA), respectively, and used for immunofluorescence. Rabbit anti-GFP antibodies were purchased from Invitrogen Molecular Probes and used for immunoblotting and immunofluorescence. Rabbit anti-FLAG antibodies were purchased from SIGMA-Aldrich (St. Louis, MO) and used for immunofluorescence and immunoblotting. Mouse monoclonal antibodies to Ubiquitin (clone P4D1; IgG1) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA).

Mouse

monoclonal antibodies to SNAP (clone MGMT-214; IgG1) were purchased from SIGMA-Aldrich. All Alexa-conjugated secondary antibodies were purchased from Invitrogen Molecular Probes (Grand Island, NY). MARCH1-FLAG, MARCH4-FLAG and MARCH8-FLAG were described previously (Eyster et al., 2011). GFP-TRE17 and GFP-TRE17/USP- are as described (Martinu et al., 2004; Shen et al., 2005). cDNA of USP8 was generously provided by Yihong Ye. USP8 and USP32 were amplified by PCR and subcloned into pEGFP-C1 (Clontech, Mountain View, CA) to generate GFP-USP8 and GFP-USP32.

GFP-USP8-Ras C20 was generated by inserting the

farnesylation signal from pEGFP-F (Clontech), which contains 20 a.a. C-terminus of c-Ha-Ras, and USP8 gene into pEGFP-C1. CD98-SNAP was described previously (Eyster et al., 2011). 20

SNAP-Tac was generated in pcDNA3.1 using the signal peptide of hen egg lysozyme (MRSLLILVLCFLPLAALG) introduced before the second amino acid of the SNAP tag (DKDCEMKR …) sequence followed by a (GGGGS)2 linker and then extracellular, TM, and cytoplasmic tail domains of Tac.


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Cell culture and transfection HeLa cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM, Lonza) with 4.5 g/l glucose, 10% fetal bovine serum, 2 mM glutamine, 100 units/ml penicillin, and 100 g/ml streptomycin and grown in a 5% CO2 atmosphere at 37 °C. Beas2B cells were cultured as HeLa cells except for glucose concentration of 1.0 g/l. For transfections, HeLa or Beas2B cells were grown on coverslips (for immunofluorescence) or six-well dishes (for pull-down) and transfected using X-tremeGENE, Fugene 6 (both from Roche Diagnostics, Indianapolis, IN), or Lipofetamine 2000 (LifeTechnologies, Grand Island, NY) according to manufacturers’ instructions. Transfections with multiple plasmids were performed with equal amount of each plasmid. Experiments were performed 20-24 h after transfection.

Antibody internalization and immunofluorescence HeLa cells or Beas2B cells were plated on glass coverslips 2 days prior to use.

The next day,

cells were transfected and further cultured for 20-24 h. For antibody internalization experiments, cells were preincubated with primary antibodies to MHCI (5 g/ml), CD44 (2.5 g/ml), or CD98 (2.5 g/ml) for 1 h at 37 ºC and then transferred to fresh media containing 25 mM NH4Cl for 2 h.

Cells were fixed for 10 min in 2% formaldehyde in PBS, rinsed with PBS,

and incubated with primary antibodies in PBS containing 10% fetal bovine serum with 0.2% saponin.

Alexa-dye conjugated secondary antibodies were used to detect the primary 21

antibodies. All images were obtained using 510 LSM (Zeiss, Thornwood, NY), FV10i-LIV (Olympus, Tokyo, Japan), and TCS-SP5 (Leica Microsystems, Wetzlar, Germany) confocal microscopes. To quantify the effect of DUB proteins, 50-60 cells expressing both MARCH8 and GFP-tagged proteins were selected and the number of cells that their cargo proteins were down-regulated and/or accumulated at a juxtanuclear compartment were counted. In


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experiments that follow the long-term fate of surface proteins, HeLa cells were preincubated with anti-MHCI antibodies at 4 ºC for 1 h to label the surface MHCI, washed with media, and further incubated at 37 ºC for 20 h. Cells were fixed and incubated with anti-mouse IgG2a Alexa 594 without saponin to detect the surface MHCI-antibody complex. Then transfected GFP-proteins were detected by anti-GFP antibody as described earlier. Images of the surface anti-MHCI antibodies were obtained using a 510 LSM confocal microscope with the pinhole completely opened. To quantify the surface level of MHCI, 40-50 transfected cells were selected and the fluorescence signals were quantified with MetaMorph (Molecular Devices, Sunnyvale, CA). The value of each cell was normalized to the cell area. To calculate the fold increase from the control (GFP-transfected cells), the mean value of the GFP control was calculated and then intensity of each cell was normalized to the mean value of GFP control and plotted.

Pull-down and immunofluorescence of SNAP-tagged proteins For pull-down experiments, HeLa cells (6-well plate) were transfected with CD98-SNAP or SNAP-Tac and GFP-tagged proteins with or without MARCH8-FLAG. After 24 h, cells were labeled with BG-PEG4-Biotin substrate (3 mM) for 4 h at 37 ºC. Cells were rinsed three times with PBS, lifted, and pelleted at 300 x g. Cell pellets were solubilized in 50 l of lysis buffer [50 mM Tris-HCl, pH 7.4, 0.25 M NaCl, 0.1% Triton X-100, 1 mM EDTA, 1 mM dithiothreitol 22

and Complete Protease Inhibitor tablets (Roche Diagnostics, Indianapolis, IN)] containing 400 M BG-NH2 and incubated on ice for 15 min. The cell extract was denatured by adding SDS to the final concentration of 1% and boiled for 15 min. The SDS was quenched by adding 50 l of 10% Triton-X100 and 450 l of lysis buffer without protease inhibitors and placed on ice for 30 min.

The lysate was centrifuged at 13,000 x g for 5 min, and 12 l of a 1:1 slury of


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streptavidin-agarose (SIGMA-Aldrich) was added to the supernatant.

The lysate was rocked at

room temperature for 1 h, and the beads were washed three times with lysis buffer without protease inhibitors. The beads were boiled for 10 min with 2 x sample buffer to elute the bound proteins. The eluate was separated by SDS-PAGE (4-12% Tris-glycine gel; Invitrogen), transferred to nitrocellulose membrane and immunoblotted with specified antibodies. The intensities of anti-ubiquitin blot and anti-SNAP blot of pulled-down fraction were quantified with Odyssey Imaging System (LI-COR, Lincoln, NE). For immunofluorescence, HeLa cells were incubated with DMEM containing BG-Alexa 594 to label the SNAP-tagged proteins at 37 ºC for 1 h. Cells were than washed and transferred to fresh media containing 25 mM NH4Cl for 2 h. Cells were stained as describe earlier.

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Acknowledgements The authors would like to thank C. Williamson and D. Karabacheva for comments on the manuscript and the Donaldson lab group for discussions. We also thank Nelson Cole (NHLBI) for the SNAP-tag constructs and Yihong Yi (NIDDK) for USP8 construct.

Microscopes used


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were in the NHLBI Light Microscopy Core Facility.

This work was supported by the Intramural Research Program in the National Heart, Lung, and Blood Institute at the National Institutes of Health (HL006060).

Author Contributions YF, MMC, YK and JGD planned the experiments; YF executed the experiments; YF and JGD interpreted the data and prepared the draft manuscript; YF, MMC, YK and JGD edited the manuscript.

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Figure Legends

Fig. 1 TRE17 counteracts the MARCH8-mediated targeting of CIE cargo proteins to late endosomes. HeLa cells were transfected with MARCH8-FLAG and GFP, GFP-TRE17 wild type or


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GFP-TRE17/USP- (DUB mutant). After 24 h, anti-MHCI (A) or anti-CD98 (C) antibodies were added to cells and allowed to bind to and be internalized for 1 h. Cells were washed and transferred to media containing 25 mM NH4Cl for 2 h. Cells were then fixed, and the internalized anti-MHCI (A) and anti-CD98 (C) antibodies were detected with anti-mouse IgG2a Alexa 594 (red) and anti-mouse IgG Alexa 594, respectively. GFP-tagged proteins were immunolabeled in (A) with mouse anti-GFP and anti-mouse IgG1 Alexa488 (green). In (C), GFP-tagged proteins were directly detected by their fluorescence. MARCH8-FLAG was detected by rabbit anti-FLAG and anti-rabbit IgG Alexa633 (blue). Cells co-expressing MARCH8 and GFP-tagged proteins are outlined by dashed lines. Arrows indicate the cells expressing MARCH8 alone. Bars, 10 m. Quantification results of (A) and (C) are shown in (B) and (D), respectively. 50-60 cells were randomly selected from transfected cells and the number of cells showing the effect of MARCH8 (reduced surface and perinuclear accumulation) was counted and plotted as % of transfected cells. Shown are means ± SEM from three independent experiments.

Fig. 2 USP8 and USP32 cannot counteract the effect of MARCH8 on CIE cargo trafficking. HeLa cells were transfected with MARCH8-FLAG and GFP-TRE17 wild type, GFP-USP8 or GFP-USP32. After 24 h, anti-MHCI (A) or anti-CD98 (C) antibodies were added to cells and 32

allowed to internalize for 1 h. Cells were washed and transferred to media containing 25 mM NH4Cl for 2 h. Antibodies for MHCI (A) and CD98 (C), GFP-tagged proteins and MARCH8-FLAG were detected as in Figure 1. Cells co-expressing MARCH8 and GFP-tagged proteins are outlined by dashed lines.

Arrows indicate the cells expressing MARCH8 alone.

Bars, 10 m. Cells were counted as in Figure 1 and the result are shown in (B) and (D).


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Shown are means ± SEM from three independent experiments.

Fig. 3 TRE17 does not counteract the MARCH8-mediated down-regulation of TfR. (A) HeLa cells were transfected with either GFP or GFP-TRE17 with or without MARCH8-FLAG. After 24 h, cells were fixed and labeled with rabbit anti-FLAG and mouse anti-transferrin receptor, followed by immunofluorescence with anti-rabbit Alexa 633 (blue) and anti-mouse Alexa 594 (red). Arrows indicate the cells co-expressing MARCH8 and GFP-tagged proteins. Bar, 20 m. (B) 50-60 cells were randomly selected from transfected cells and the number of cells showing the effect of MARCH8 (down-regulation TfR) was counted and plotted as % of transfected cells. Shown are means ± SEM from four independent experiments.

Fig. 4 TRE17 colocalizes with MHCI and CD98 but not with TfR. HeLa cells were transfected with GFP-TRE17 and cultured for 24 h. Cells were fixed and incubated with rabbit anti-GFP and anti-MHCI, CD98, or TfR antibodies as indicated, followed by secondary antibodies, anti-rabbit IgG Alexa488 (green) and anti-mouse IgG Alexa594 (red). Bar, 10 m.

Fig. 5 33

The USP8-Ras C20 chimera co-localizes with CIE cargo proteins but not with TfR. HeLa cells were transfected with GFP-USP8 (A) or GFP-USP8-Ras C20 (B) and cultured for 24 h.

Cells

were fixed and incubated with chicken anti-GFP and anti-MHCI, CD98, or TfR antibodies, followed by secondary antibodies, anti-chicken IgG Alexa488 (green) and anti-mouse IgG


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Alexa546 (red).

Bars, 10 m.

Fig. 6 The USP8-Ras C20 chimera counteracts the MARCH8-mediated targeting of CIE cargo proteins to late endosomes but not the down-regulation of TfR. (A-D) HeLa cells were transfected with MARCH8-FLAG and GFP-TRE17 (not shown in A and C), GFP-USP8 or GFP-USP8-Ras C20. After 24 h, anti-MHCI (A and B) or anti-CD98 (C and D) antibodies were added to cells and treated as in Figure 1. Cells were then fixed, and the internalized anti-MHCI (A and B) and anti-CD98 (C and D) antibodies were detected with anti-mouse IgG Alexa 546 (red). GFP-tagged proteins were immunolabeled with chicken anti-GFP and anti-chicken IgG Alexa488 (green). MARCH8-FLAG was detected by rabbit anti-FLAG and anti-rabbit IgG Alexa633 (blue). Cells co-expressing MARCH8 and GFP-tagged proteins are outlined by dashed lines. Arrows indicate the cells expressing MARCH8 alone. Bars, 20 m. The cell number was counted and plotted in (B) and (D) as in Figure 1. Shown are means ± SEM from three independent experiments. (E and F) HeLa cells were transfected with MARCH8-FLAG and GFP-TRE17 (not shown in E), GFP-USP8 or GFP-USP8-Ras C20. After 24 h, cells were fixed and labeled with chicken anti-GFP, rabbit anti-FLAG and mouse anti-transferrin receptor, followed by immunofluorescence with anti-chicken Alexa 488 (green), anti-rabbit Alexa 633 (blue) and anti-mouse Alexa 546 (red). The cell number was counted and plotted in (F) as in Figure 3. Shown are means ± SEM from three independent experiments. In (A), (C) and (E), 34

cells co-expressing MARCH8 and GFP-tagged proteins are outlined by dashed lines. Arrows indicate the cells expressing MARCH8 alone. Bars, 20 m.

Fig. 7 TRE17 deubiqutinates CD98 and Tac. (A) HeLa cells were transfected with CD98-SNAP and


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GFP, GFP-TRE17 wild type, GFP-TRE17/USP-, GFP-USP8 or GFPUSP32 with or without MARCH8-FLAG as indicated. After 24 h, CD98-SNAP was labeled with BG-biotin and pulled-down by streptavidin agarose from denatured cell lysates as described in Materials and Methods. Cell lysates and precipitates were separated on SDS-PAGE and immunoblotted with antibodies as indicated. (B) Quantification of Ubiquitinated proteins in (A). The intensities of ubiqutinated CD98-SNAP that are indicated by the bracket in anti-ubiquitin blot were measured and normalized to the respective precipitated CD98-SNAP. The values were plotted as fold increase from control cells (GFP without MARCH8). Shown are means ± SEM from four independent experiments.

*: P < 0.05 and n.s.: not significant compared with cells transfected

with GFP and MARCH8 by one-way ANOVA with post hoc Dunnett’s test.

(C) HeLa cells

were transfected with SNAP-Tac and GFP, GFP-TRE17 wt, GFP-TRE17/USP-, GFP-USP8 or GFPUSP32 with or without MARCH8-FLAG as indicated. After 24 h, SNAP-Tac was labeled, pulled-down and immunoblotted as in (A). (D) Ubiquitinated SNAP-Tac in (C) was quantified and plotted as in (B). Shown are means ± SEM from three independent experiments. **: P < 0.005 and n.s.: not significant compared with cells transfected with GFP and MARCH8 by one-way ANOVA with post hoc Dunnett’s test.

Fig. 8

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TRE17 expression alone increases cell surface MHCI. (A) HeLa cells were transfected with GFP, GFP-TRE17 wild type or GFP-TRE17/USP-.

After 24 h, cells were pre-incubated with

anti-MHCI antibodies at 4 ºC for 1 h to label the cell surface MHCI. Cells were washed and further incubated at 37 ºC for 0 h or 20 h. Cells were then fixed and incubated with anti-mouse IgG2a Alexa 594 without saponin to label the cell surface anti-MHCI antibodies. GFP-tagged


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proteins were detected by using mouse anti-GFP and anti-mouse IgG1 Alexa 488. Images of 0 h and 20 h were obtained with individual settings of a confocal microscope. Bars, 20 m. (B) Quantification of cell surface MHCI in (A). 40-50 cells were randomly selected from transfected cells and the intensity of fluorescence signals of the surface MHCI were measured and normalized to each cell area. The values were plotted as fold increase from GFP-expressing cells in each time point (0 h and 20 h). Shown are means ± SD for one representative experiment, repeated one additional time. **: P < 0.005 and n.s.: not significant compared with GFP-expressing cells by one-way ANOVA with post hoc Dunnett’s test.

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