Adaptor linked K63 di-ubiquitin activates Nedd4/Rsp5 E3 ligase

Nedd4/Rsp5 family E3 ligases mediate numerous cellular processes, many of which require the E3 ligase to interact with PY motif containing adaptor proteins. Several arrestin-related trafficking adaptors (ARTs) of Rsp5 were self-ubiquitinated for activation, but the regulation mechanism remains elusive. Remarkably, we demonstrate that Art1, Art4, and Art5 undergo K63-linked di-ubiquitination by Rsp5. This modification enhances the plasma membrane recruitment of Rsp5 by Art1 or Art5 upon substrate induction, required for cargo protein ubiquitination. In agreement with these observations, we find that di-ubiquitin strengthens the interaction between the pombe orthologs of Rsp5 and Art1, Pub1, and Any1. Furthermore, we discover that the homologous to E6AP C-terminus (HECT) domain exosite protects the K63-linked di-ubiquitin on the adaptors from cleavage by the deubiquitination enzyme Ubp2. Together, our study uncovers a novel ubiquitination modification implemented by Rsp5 adaptor proteins, underscoring the regulatory mechanism of how adaptor proteins control the recruitment, and activity of Rsp5 for the turnover of membrane proteins.


Introduction 24
The Nedd4/Rsp5 family E3 ligases are responsible for membrane protein ubiquitination, required 25 for endocytosis and lysosome-dependent protein degradation. Tryptophan-tryptophan (WW) 26 domains of Nedd4 family E3 ligases bind to substrate proteins via interaction with PY motifs 27 containing a consensus sequence P/L-P-x-Y (Rotin & Kumar, 2009;Schild et al, 1996). Other 28 substrates lack PY motifs and instead rely on interactions with adaptor proteins that recruit the 29 Nedd4 E3 ligase to them, exemplified by a family of arrestin-related trafficking adaptors (ARTs) 30 that bridge the association between substrates and Rsp5 for ubiquitination (Lin et al, 2008). Since ARTs ubiquitination enhances function, we next sought to test if C-terminal fusion with 184 ubiquitin molecules could rescue the cargo sorting defect of art1 K486R or art1 ∆PY . Since toxic 185 arginine analog canavanine is transported by PM transporter Can1 in yeast and Can1 endocytosis 186 prevents subsequent cell death (Grenson et al, 1966), canavanine hypersensitivity occurs when 187 Can1 cannot be endocytosed (such as in an art1∆ mutant), which provides a readout of Art1 188 function. Thus, we examined the canavanine sensitivity of the art1∆ mutant expressing Art1 WT , 189 art1 K486R or art1 ∆PY fused with C-terminal 1x, 2x or 3xUb. The C-terminal fusions with ubiquitin 190 molecules did not enhance the functionality of Art1 WT , art1 K486R or art1 ∆PY (Fig. S2I, S2J). Besides 191 Art1, we tested if Itr1-GFP sorting can be restored by art5 K364R with C-terminal 1xUb or 2xUb and 192 found the 1x or 2xUb fusions do not enhance the Itr1 sorting (Fig. 2F). Together, our data indicate 193 that di-ubiquitin needs to be conjugated at specific residues for proper functionality. The art5 K364R mutant partially blocks the ubiquitination and cargo sorting of Itr1 after inositol 197 treatment (Figure 2A and 2D), but still interacts with Itr1. We therefore hypothesize that the 198 defective ubiquitination of art5 K364R may impair Rsp5 recruitment to the PM. To test this idea, we 199 examined the localization of Art5-GFP in yeast cells before and after inositol treatment. As seen 200 in figure 3A, the Art5 WT -GFP localized at cytosol, nucleus and occasional cytosolic puncta (Sec7-201 negative, Figure 3A). Strikingly, the Art5 WT -GFP is re-localized to PM puncta and patch structures 202 after 1 hour of inositol (20µg/ml) treatment. This result is in line with our previous finding that 203 Art5 is partially translocated to peripheral puncta after shift from minimal media to YPD (Lin et 204 al., 2008), probably because the inositol concentration in YPD is not high enough to drive 205 significant Art5 re-localization and Itr1 endocytosis. In comparison to Art5 WT , the art5 K364R -GFP 206 and art5 ∆PY mainly remain in the cytosol even after inositol treatment ( Figure 3B-D). We conclude 207 that ubiquitination of Art5 is important for protein re-localization to the PM upon inositol treatment. 208 We next asked if Rsp5 can be re-localized to the PM in an Art5-dependent manner after adding 209 inositol to the growth media. As expected, Rsp5 was observed to be recruited to PM patches after 210 inositol treatment in WT cells. However, Rsp5 PM recruitment after inositol treatment was 211 substantially reduced in cells expressing either art5 K364R or art5 ∆PY (Figure 3E and 3F (Lin et al., 2008). As seen in the figure S3A, Art1 is efficiently recruited 218 to the PM in YPD or in methionine media. In contrast to Art1 WT , the recruitment of art1 K486R to 219 the PM is largely attenuated and no PM recruitment is seen with art1 ∆PY (Figure S3B, S3C) . We 220 next tested whether Art1 facilitates PM recruitment of Rsp5. As expected, methionine treatment 221 induces Rsp5 PM recruitment in cells expressing WT Art1, but this recruitment is much reduced 222 in cells expressing art1 K486R ( Figure S3E, S3F). Taken together, our results support the model that 223 specific ubiquitination of adaptor proteins is required for proper recruitment of Rsp5 to target 224 membranes and subsequent ubiquitin-mediated endocytosis of cargo proteins. 225 226 Substrate dependent PM recruitment of adaptor protein Art5 and Art1 227 Next, we sought to examine if cargo proteins are required for adaptor protein recruitment to their 228 functional locations. To do so, we examined the Art5-GFP PM recruitment in ITR1-WT and itr1∆ 229 mutants upon inositol treatment for 1 hour. Strikingly, we found that the PM recruitment of Art5-230 GFP is abolished in the itr1∆ mutant ( Figure 3G, 3H). Similarly, we observed that the PM 231 recruitment of Art1 is attenuated in the mup1∆ mutant with methionine induction for 1 hour (figure 232 S3G and S3H). We further tested whether Art1 can be recruited to the PM in cells expressing 233 mup1-Q49R mutant upon methionine treatment. Previous data showed that Mup1 mutant Q49R 234 is unable to be endocytosed with methionine treatment (Guiney et al., 2016) and the mup1-Q49R 235 mutation abolishes the protein-protein interaction between Mup1 and Art1 ( Figure S2E). We 236 expected that PM recruitment of Art1 will be impaired in this mutant due to the loss of the 237 interaction between Art1 and Mup1-Q49R. Indeed, PM recruitment of Art1 is abrogated in the 238 mup1-Q49R condition ( Figure S3G and S3H), suggesting that the Mup1-Art1 interaction is 239 required for methionine-induced Art1 PM re-localization. Collectively, our data demonstrate that 240 the substrate proteins (Itr1 and Mup1) are required for adaptor protein (Art5 and Art1) recruitment 241 to target membranes (Fig. 3I). 242

K63-linked di-ubiquitination enhances the interaction between adaptor proteins and Rsp5 244
We found that the Rsp5 adaptor proteins (Art5, Art1, and Art4) undergo K63-linked di-245 ubiquitination and this modification is required for efficient recruitment of Rsp5 to target 246 membranes and cargo protein degradation. We hypothesized that adaptor di-ubiquitination 247 enhances protein-protein interactions between di-ubiquitinated adaptors and Rsp5 and thus 248 promotes the recruitment of the E3 ligase. To test this hypothesis, we set out to examine the binding 249 between mono-Ub or K63-linked di-Ub and the HECT domain of Rsp5, as well as the binding 250 between PY motifs and WW domains. To do so, we first generated K63-linked ubiquitin chains showed that M1 linked di-Ub binds to HECT with Kd=36µM, comparable with the K63-linked di-283 Ub (Fig. 4D). In line with this result, our in-vitro binding assay showed that the binding between 284 GST-2xUb and HECT domain is stronger than GST-Ub (Fig. 4E). In comparison, K48-linked di-285 Ub shows a much lower affinity than K63-di-Ub, Kd=145µM (Fig. 4F). Together, our results 286 demonstrate that HECT domain specifically binds to linear form K63 di-Ub and the exosite site is 287 required for ubiquitin binding. di-Ub cannot be made (Tsui et al, 2005). We found that the K63-linked Ub I44A -Ub WT binds to 296 HECT with an Kd=120µM, lower binding affinity than the K63 di-Ub (Fig. 4G). Thus, our result 297 suggests that both distal and proximal ubiquitins contribute to the HECT domain binding, probably 298 cooperatively. 299

300
We next sought to determine if K63-linked di-Ub enhances the binding between adaptor and 301 HECT type E3 ligase. In spite of the fact that KR mutants of Art5 and Art1 lead to attenuated 302 Rsp5 PM recruitment and cargo proteins (Itr1 and Mup1), we still observed the interaction between 303 Art1-K486R or Art5-K364R with Rsp5 using Co-IP (Fig. 4H, 4I), probably due to the interaction 304 between the PY motifs and WW domains. Indeed, as shown in the figure (Fig. 4J, 4K), Art1 and 305 Art5 PY motif containing peptides interact with purified WW domains from Rsp5 (Kd= 3.6µM 306 for Art1 PY motifs and Kd=3.1µM for Art5 PY motifs), but not with PY motif mutants. Since the 307 sole interaction between PY motifs and WW domains does not suffice the full activation of Rsp5 308 function ( Fig. 2A and S2A), the interaction between di-Ub and HECT may enhance the binding 309 affinity between Need4/Rsp5 E3 ligases and their adaptors. We next sought to test the binding 310 between full length adaptors and Rsp5. We found that we could not express Art1 or Art5 at high 311 levels in E. coli, then tried to express the Art1 orthologue from S. pombe, Any1, in E.coli. We 312 found the S. pombe Rsp5 ortholog Pub1 interacts with Any1 with a binding affinity Kd~2.1µM 313  We then sought to uncover the mechanism by which the Art5 degradation is triggered. We 364 observed that Art5 protein is also degraded in the Ub-K63R mutant (Fig. 1C). We wondered if 365 ubp2∆ can rescue Art5 degradation in the Ub-K63R mutant. To test this, we deleted Ubp2 in the 366 Ub-K63R mutant and found that ubp2∆ does not reverse the loss of Art5 protein (Fig. 5D). Our 367 results suggest that the loss of K63-di-Ub on Art5, instead of Ubp2, in either the Ub-K63R mutant 368 or rsp5-exosite mutant, leads to Art5 degradation. Remarkably, in these two conditions, the Art5 369 degradation can be rescued in art5-K364R and art5-∆PY mutant ( Fig. 5B and 5D). These data 370 suggest that Rsp5 can mediate both K63 and K48-linked ubiquitination and the K364 residue of 371 Art5 can be conjugated with both K63-linked di-Ub and K48-linked polyubiquitin chain. 372 Collectively, our results support a working model that the K63-linked di-Ub on Art5 is fully 373 engaged into the Rsp5 exosite so that Ubp2 cannot cleave it efficiently, whereas the K63-linked 374 di-Ub is disengaged in an rsp5-exosite mutant therefore exposed to Ubp2 for cleavage. Upon the 375 cleavage of K63-linked di-Ub, a K48-linked polyubiquitin chain is conjugated at the same residue 376 Art5-K364 thereby leading to the proteasome-dependent degradation of Art5 (Fig. 5E-5H). 377

378
Since PM recruitment of Rsp5 is enhanced by Art5 or Art1 protein ubiquitination (Fig. 3E, S3E) 379 and Ubp2 can deubiquitinate these adaptor proteins ( Fig. 5A and S4D), we therefore asked if the 380 adaptor ubiquitination process is reversible and Ubp2 is involved in this process or not. To monitor 381 the pre-existing adaptor proteins, we decided to employ the tet-Off system to fix the pool of adaptor 382 proteins by treating the cells with doxycycline. As seen in the figure S4F and S4G, pre-existing 383 Art5 or Art1 undergoes ubiquitination upon inositol or methionine treatment for 1 hour in both 384 WT and ubp2∆ conditions, whereas the adaptor proteins shifted back to less ubiquitinated status 385 after removing the stimulation in the WT condition, but not in the ubp2∆ mutant. Together, our 386 data demonstrate a model for adaptor protein recycling mediated by Ubp2 (Fig. S4H). First, 387 stimulation enhances adaptor ubiquitination. Second, the ubiquitinated pool of adaptor proteins 388 can be de-ubiquitinated by Ubp2 when stimulation is terminated.

K63-linked di-Ub is engaged into Rsp5 E3 ligase for activation 409
While we showed that Rsp5 adaptors Art1, Art4 and Art5 undergo K63-linked di-Ub modification, 410 we also demonstrate that this conjugation sharply enhances the binding with the E3 ligase and 411 activates the E3 ligase function for substrate ubiquitination (Fig. 2D). We reason that the 412 interaction between the di-Ub chain and the HECT domain locks the E3 ligase and adaptor into an 413 active/functional conformation. For adaptor-independent ubiquitination, the Nedd4/Rsp5 ligase 414 exosite is also required for efficient ubiquitin conjugation, demonstrating that the "Ub-exosite 415 binding" is required to localize and orient the distal end ubiquitin chain to promote conjugation 416 (Kim et al., 2011;Maspero et al., 2011). In terms of the Rsp5 adaptor-mediated function, we 417 propose that the binding between "di-Ub and exosite" not only enhances the binding affinity 418 between the E3 ligase and adaptor (Fig. 4L-4M), but also leads to more productive Rsp5 419 recruitment to properly orient and present the substrate for ubiquitination at target membranes (Fig.  420   3B). 421

422
While we presented the evidence of E3 ligase activation by ubiquitinated adaptors, we also showed 423 that K63 di-Ub generates a 6-fold tighter binding to the HECT domain than mono-Ub. We reason 424 that the K63 di-Ub provides alternative options to bind a single site, but also fits with a model in of Rsp5 via various adaptors leads us to hypothesize that activated Rsp5 can be released from 477 engaged adaptor proteins. We showed that the adaptor proteins Art1 and Art5 undergo di-478 Ubiquitination upon environmental stimulation and Ubp2 is required to reverse this ubiquitination. 479 Once the ubiquitination is done, the engaged K63 di-Ub is exposed for cleavage by Ubp2. 480 Thereafter, Ubp2 acts on ubiquitinated adaptor proteins to release the adaptor proteins and Rsp5. 481 The mechanism by which Ubp2 executes this reaction needs to be addressed in the future.