Complementary α-arrestin - Rsp5 ubiquitin ligase complexes control selective nutrient transporter endocytosis in response to amino acid availability

How cells adjust transport across their membranes is incompletely understood. Previously, we have shown that S.cerevisiae broadly re-configures the nutrient transporters at the plasma membrane in response to amino acid availability, through selective endocytosis of sugar- and amino acid transporters (AATs) (Müller et al., 2015). A genome-wide screen now revealed that Art2/Ecm21, a member of the α-arrestin family of Rsp5 ubiquitin ligase adaptors, is required for the simultaneous endocytosis of four AATs and induced during starvation by the general amino acid control pathway. Art2 uses a basic patch to recognize C-terminal acidic sorting motifs in these AATs and instructs Rsp5 to ubiquitinate proximal lysine residues. In response to amino acid excess, Rsp5 instead uses TORC1-activated Art1 to detect N-terminal acidic sorting motifs within the same AATs, which initiates exclusive substrate-induced endocytosis of individual AATs. Thus, amino acid availability activates complementary α-arrestin-Rsp5-complexes to control selective endocytosis for nutrient acquisition.


Introduction
10 et al., , Schuldiner et al., 1998. Disrupting the GAAC pathway (gcn4∆) eliminated the induction 275 of Art2 in response to starvation at mRNA and protein levels (Fig. 4B,C). Consistently, the starvation-276 induced endocytosis of Mup1-GFP was hampered in gcn4∆ cells and several other gcn mutants (Fig. 277 4D,S4B). Also, starvation-induced endocytosis of Can1 was dependent on the GAAC pathway (Fig. 278 S4C). When we introduced mutations in the predicted Gcn4 binding sites in the ART2 promoter, Art2 279 protein levels no longer increased in response to starvation, and starvation-induced endocytosis of 280 Mup1-GFP was impaired (Fig. 4E). 281

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The expression of a constitutively translated Gcn4 C construct (Mueller and Hinnebusch, 1986) increased 283 Art2 protein levels already under rich conditions, as revealed by WB analysis (Fig. 4F, compare Art2 284 protein levels in lanes 1 and 3), and drove unscheduled Mup1-GFP endocytosis (Fig. 4F, lower panel). 285 Consistently, over-expression of Art2 in WT cells or in gcn4∆ mutants using the strong and 286 constitutively active TDH3 promoter ( To determine how Art2 contributed to Mup1 endocytosis, we examined its role in Mup1 ubiquitination. 296 WT cells were harvested and Mup1-GFP was immunoprecipitated in denaturing conditions before and 297 at different time points after starvation. Equal amounts of immunoprecipitated full-length Mup1-GFP 298 were subjected to SDS-PAGE and WB analysis to compare the extent of its ubiquitination at different 299 time points (Fig 5A). This analysis indicated that a pool of Mup1 was ubiquitinated prior to the onset of 300 starvation (Fig. 5A, lane 1). At the onset of starvation, ubiquitination of Mup1-GFP appeared to decrease 301 for some time (Fig. 5A lanes 2-4), until ubiquitination of Mup1-GFP began to increase again after 2-3 302 hours during starvation ( Fig. 5A lanes 4-6), temporally coinciding with Art2 induction and starvation-303 induced endocytosis. Mup1-GFP was still ubiquitinated in art2D cells growing under rich conditions 304 ( Fig. 5B lane 4) and seemingly de-ubiquitinated at the onset of starvation, but the increase of 305 ubiquitination during starvation was no longer observed (Fig. 5B, lane 6). Hence, Art2 was essential for 306 the starvation-induced ubiquitination of Mup1. 307 308 α-Arrestins use PY motifs to bind to at least one of the three WW domains of Rsp5 (Lin et al., 2008). 309 Starvation-induced endocytosis of Mup1 (but not methionine-induced endocytosis) was particularly 310 dependent on the WW3 domain of Rsp5 (Fig. 5C, D, S5A). Art2 has four putative PY motifs (Fig. 5C) 311 and the PY motif within the predicted arrestin fold of Art2 (P748,P749,Y750) was required for 312 starvation-induced endocytosis (Fig. 5E). The Art2 P748A,P749A,Y750A mutant was expressed at similar levels 313 than the WT protein (Fig. S5B). We suggest that interaction between WW3 in Rsp5 and the PY motif 314 (748-750) of Art2 was required for the starvation-induced endocytosis of Mup1. 315

378
The C-terminus of Mup1 also contains an acidic patch (D549-D555), close to the ubiquitination sites 379 K567 and K572 and the C-terminal threonine phosphorylation sites (T552, 560) involved in starvation-380 induced endocytosis (Fig. 6A, B). Mutation of the acidic residues in this region to basic amino acids (R) 381 demonstrated that this C-terminal acidic region was specifically required for starvation-induced 382 endocytosis. Live cell fluorescence microscopy revealed that Mup1 D549R,D551R,E554R,D555R -GFP remained 383 at the PM in response to starvation, whereas methionine-induced endocytosis was not impaired (Fig. 384 6B). Even Art2 overexpression failed to induce endocytosis during exponential growth or starvation 385 13 when the C-terminal acidic patch in Mup1 was mutated (Fig. S6B). Moreover, immunoprecipitation of 386 Mup1 D549R,D551R,E554R,D555R -GFP and subsequent SDS-PAGE and WB analysis revealed that it was no 387 longer efficiently ubiquitinated (Fig. 6C, lanes 4 -6), suggesting that the C-terminal acidic patch was 388 essential for the Art2-Rsp5-dependent ubiquitination during starvation. 389 390 Comparing the amino acid sequences of the C-terminal tails of the four Art2-dependent cargoes, Mup1, 391 Can1, Tat2 and Lyp1, indicated similar acidic patches (Fig. 6D). To analyze if the acidic patch in Can1 392 also contributed to starvation-induced endocytosis, we mutated D567, E569, E574 and E575 to arginine. 393 Mutant Can1 D567R,E569R,E574R,D575R -GFP mostly localized to the PM under growing conditions. 394 Importantly, the Art2-dependent starvation-induced down-regulation of Can1 D567R,E569R,E574R,D575R -GFP 395 was impaired (Fig. S6C). These results imply that Mup1, Can1 and potentially also Lyp1 and Tat2 have 396 acidic amino acid sequences at their C-termini that could serve as sorting signal for Art2-mediated 397 starvation-induced endocytosis. 398

399
The C-terminal acidic sorting signal of Mup1 is sufficient for Art2-dependent starvation-induced 400 endocytosis 401 It seemed that the last 26 amino acid residues (aa 549-574) of Mup1 harbor three features that are 402 collectively required specifically for starvation-induced endocytosis: putative phosphorylation sites, the 403 acidic patch and the ubiquitination sites. Hence, we tested if the C-terminal region of Mup1 was 404 sufficient to convert an Art2-independent cargo into an Art2 cargo. We selected the low affinity glucose 405 transporter Hxt3, which was efficiently removed from the PM in response to starvation (Fig. S6D, 406 Table S1). Live cell fluorescence microscopy and WB analysis showed that starvation-induced 407 endocytosis of Hxt3-GFP was independent of Art2, but instead required Art4 (Fig. S6D). In art4∆ 408 mutants, but not in art2D mutants, Hxt3-GFP remained mostly at the PM ( The Art2-dependent endocytosis of the Hxt3-Mup1-C-GFP chimera required two key features provided 422 14 by the C-terminus of Mup1 (the acidic patch and the two C-terminal lysine residues), since in art4∆ 423 cells starvation-induced endocytosis of Hxt3-Mup1-C K567,572R -GFP and Hxt3-Mup1-424 C D549R,D551R,E554R,D555R -GFP was blocked (Fig. 6E). 425 426 Taken together, these results demonstrate that the C-terminus of Mup1 (aa 545-574) encodes a portable 427 acidic sorting signal that can be recognized by Art2 and directs Rsp5 to ubiquitinate specifically two 428 proximal lysine residues to promote starvation-induced endocytosis. 429

A basic patch of Art2 is required for starvation-induced degradation of Mup1 431
After having defined that the C-terminus of Mup1 (and possibly also the C-terminus of the AATs Can1, 432 Lyp1 and Tat2) provides a degron sequence for Art2-Rsp5 complexes, we addressed how it could be 433 specifically recognized. Upon inspection of the predicted arrestin domain in Art2, we noted a stretch of 434 positively charged residues within the arrestin-C domain (Fig. 7A). Converting these basic residues into 435 an acidic patch (Art2 K664D,R665D,R666D,K667D ) abolished starvation-induced endocytosis of Mup1 (Fig. 7B). 436 Western blot analysis of total cell lysates showed that the Art2 basic patch mutant protein was expressed 437 at similar levels as WT Art2 and was also upregulated after 3 hours of starvation (Fig. S7A, lane 6). In 438 addition, the Art2 basic patch mutant also impaired, at least partially, starvation-induced endocytosis of 439 Can1 and Lyp1, while the endocytosis of Tat2 was independent of the basic patch (Fig. 7B, S7B). defined PY motifs to orient Rsp5 with high specificity towards proximal lysine residues. These rules 459 satisfy the plasticity required for different α-arrestin and AAT interactions that drive exclusive or 460 relatively broad substrate specificity depending on the metabolic context. 461 462 While both Art1 and Art2 lead to the degradation of AATs, they answer to distinct metabolic cues and 463 are thus wired into distinct signaling pathways. Activation of Art1 by amino acid influx requires the 464 coordinated interplay of TORC1 signaling to inactivate Npr1 (a kinase that negatively regulates Art1) 465 and the action of phosphatases (Gournas et al., 2017, Lee et al., 2019, MacGurn et al., 2011, Tumolo et 466 al., 2020. In response to amino acid limitation, TORC1 is no longer active. This will activate Npr1 to 467 phosphorylate Art1, thereby inactivating it. At the same time, the lack of amino acids will activate the 468 eIF2a kinase Gcn2. Gcn2 will phosphorylate eIF2a, which leads to the global down-regulation of 469 translation, but enables specific translation of the transcription factor Gcn4 (Hinnebusch, 2005). Gcn4 470 then induces transcription of genes required for amino acid biosynthesis and of ART2, which causes an 471 increase in Art2 protein levels and thus formation of Art2-Rsp5 complexes. This appears as primary 472 means to activate Art2, since unscheduled increase in Art2 protein levels was sufficient to drive Art2-473 dependent nutrient transporter endocytosis already in cells growing under rich conditions. When amino 474 acids become available again, the system can efficiently reset. TORC1 is reactivated resulting in Art1 475 reactivation. Conversely, Gcn4 will become instable and rapidly degraded by the UPS (Kornitzer et al., 476 1994, Meimoun et al., 2000, Irniger and Braus, 2003, and thus, the transcription of ART2 will cease. 477 Interestingly, two de-ubiquitinating enzymes (Ubp2, Ubp15) de-ubiquitinate Art2 to influence its 478 protein stability (Ho et al., 2017, Kee et al., 2006. Inhibiting their activity could provide additional 479 control to repress Art2-dependent endocytosis in cells growing under rich conditions. Our screen 480 identified also two de-ubiquinating enzymes, Doa4 and Ubp6 to be specifically required for starvation-481 induced endocytosis of Mup1. They could act directly on Art2 or Mup1 or help to maintain homeostasis 482 of the ubiquitin pool during starvation. 483 16 484 Art2 is subject to extensive post-translational modification, including ubiquitination and 485 phosphorylation. Database searches and our own proteomic experiments identified 68 phosphorylation 486 sites and 20 ubiquitination sites in Art2 (data not shown) (Swaney et al., 2013, Albuquerque et al., 2008, 487 Holt et al., 2009. How these modifications help to control the activity of Art2 remains a complex and 488 open questions. Several arrestins were found to be phospho-inhibited in specific conditions (MacGurn 489 et al., 2011, Becuwe et al., 2012b, O'Donnell et al., 2013, Hovsepian et al., 2017, Merhi and André, 490 2012, Llopis-Torregrosa et al., 2016, the common molecular basis of which is unknown. An exciting 491 hypothesis would be that a-arrestin hyper-phosphorylation would add negative charges, and thereby 492 prevent the recognition of acidic patches on transporters through electrostatic repulsion. Interestingly, 493 our screen identified the pleitropic type 2A-related serine-threonine phosphatase Sit4 as a class 1 hit. 494 Hence, Sit4 may be linked directly or indirectly to de-phosphorylation of Art2 and controlling its activity 495 as reported recently for the Art2-dependent regulation of vitamin B1 transporters (Savocco et al., 2019). 496

497
Through the complementary activation of Art1 and Art2 cells can coordinate amino acid uptake through 498 at least four high-affinity amino acid transporters with amino acid availability. The regulation of hexose 499 transporters by glucose availability appears to be conceptually related, with distinct α-arrestin-Rsp5 500 complexes in charge of down-regulating the same transporters at various glucose concentrations with 501 distinct mechanisms and kinetics (Hovsepian et al., 2017, Nikko andPelham, 2009). In particular, the 502 endocytosis of high-affinity hexose transporters during glucose starvation involves Art8, the closest 503 paralogue of Art2, whose expression is also controlled by nutrient-regulated transcription (Hovsepian 504 et al., 2017). Altogether, a picture emerges in which the transcriptional control of α-arrestin expression 505 by nutrient-signaling pathways is critical to cope with nutrient depletion. 506 507 Our work also extends on previous findings regarding the determinants of α-arrestin/transporter 508 interaction, indicating communalities between starvation-and substrate-induced endocytosis. Art1-509 Rsp5 and Art2-Rsp5 complexes both recognize specific acidic sequences on Mup1 ( conformation, which in Mup1 also includes the so-called 'C-plug' (aa 520-543) (Busto et al., 2018, 514 Guiney et al., 2016, Gournas et al., 2017. This conformational switch drives lateral re-localization of 515 Mup1 and Can1 into a disperse PM compartment, where they are ubiquitinated by Art1-Rsp5 (Gournas 516 et al., 2018, Busto et al., 2018. Art2 recognizes specifically an acidic patch in the C-terminal tail of 517 Mup1, and thereby directs Rsp5 to ubiquitinate two juxtaposed C-terminal lysine residues. The C-plug 518 is very close to the C-terminal acidic patch, but is not part of the C-terminal Mup1 degron. We speculate 519 that in the absence of nutrients AATs will spend more time in the outward open state with the C-Plug 520 in place. In this state, activated Art2-Rsp5 complexes can still engage the C-terminal acidic patches. 521 Hence, toggling Art1/Art2 activation in combination with accessibility of N-or C-terminal acidic sorting 522 signals in AATs, in part regulated by their conformational state, must fall together to allow selective 523 endocytosis. 524 525 An additional layer of regulation for endocytosis is provided by phosphorylation of AATs close to the 526 acidic sorting signal. At the moment we can only speculate about the kinase responsible for the 527 phosphorylation of the C-terminal serine or threonine sites of Mup1. Perhaps, constitutive PM-528 associated kinases such as the yeast casein kinase 1 pair (Yck1/2) are involved, which are known to 529 recognize rather acidic target sequences and to regulate endocytosis (Hicke et al., 1998, Paiva et al., 530 2009, Nikko et al., 2008, Marchal et al., 2002. 531 532 α-Arrestins lack the polar core in the arrestin domain that is used for cargo interactions in β-arrestins 533 (Aubry et al., 2009, Polekhina et al., 2013. Instead Art1-Rsp5 and Art2-Rsp5 complexes each use a 534 basic region in their arrestin C-domain to detect the acidic sorting signal in their client AATs. Studies 535 on the interaction between GPCRs and β-arrestins revealed a multimodal network of flexible 536 interactions: The N-domain of b-arrestin interacts with phosphorylated regions of the GPCR, their finger 537 loop inserts into the transmembrane domain bundle of the GPCR and loops at the C-terminal edge of b-538 arrestin engage the membrane (Staus et al., 2020, Huang et al., 2020. Perhaps a similar concept also 539 holds true for α-arrestins. This is not unlikely given that their arrestin fold appears to be interspersed 540 with disordered loops and very long, probably unstructured N-and/or C-terminal tails, some of which 541 participate in cargo recognition or membrane interactions (Baile et al., 2019). 542 543 Despite the possible plasticity in substrate interactions, the selectivity of Art1-Rsp5 and Art2-Rsp5 544 complexes in ubiquitinating lysine residues proximal to the acidic patches of Mup1 is remarkable. Mup1 545 has 19 lysine residues at the cytoplasmic side: four at the N-terminal tail, six in the C-terminal tail and 546 9 in the intracellular loops of the pore domain. Yet, Art1-Rsp5 complexes only ubiquitinate K27 and 547 K28, whereas Art2-Rsp5 complexes only ubiquitinate K567 and K572. Also in the Hxt3-Mup1-C 548 chimeric protein, Art2-Rsp5 complexes ubiquitinated only the lysine residues close to the acidic patch, 549 despite 6 further lysine residues in the directly adjacent C-terminal tail of Hxt3. How is this possible? 550 We speculate that these two α-arrestin-Rsp5 complexes orient the HECT domain of Rsp5 with high 551 precision towards the lysine residues that are spatially close to the acidic patches. Once ubiquitinated, 552 the AAT can engage the endocytic machinery to be removed from the PM. 553

554
In conclusion, Art1-Rsp5 complexes act rapidly to prevent the accumulation of excess amino acids, 555 whereas the Art2-Rsp5 complexes help to degrade idle high affinity amino acid transporters over longer 556 periods of starvation to recycle their amino acid content. Starvation-induced endocytosis and the 557 subsequent degradation of membrane proteins is required to maintain intracellular amino acid 558 homeostasis (Müller et al., 2015, Jones et al., 2012. As such, it is well suited that Art2 activity and thus 559 starvation-induced endocytosis is co-regulated and coordinated with de novo amino acid biosynthesis 560 via the GAAC pathway. The down-regulation of AATs together with glucose transporters and further 561 PM proteins could also free up domains at the PM that are populated by selective nutrient transporters 562 (Spira et al., 2012, Grossmann et al., 2008 for transporters with broader substrate specificity such as 563 the general amino acid permease Gap1 and the ammonium transporter Mep2, which are strongly up-564 regulated during starvation. Hence starvation-induced endocytosis could prepare cells -anticipatory -565 for non-selective nutrient acquisition, as soon as nutrients become available again. Yeast strains used for the microscopy screen for starvation-responsive endocytosis cargoes were mainly 578 derived from the Yeast C-terminal GFP Collection (Huh et al., 2003) with addition of further C-579 terminally-tagged transporters in BY4741 (MATa his3Δ1 leu2Δ0 met15Δ0 ura3Δ0) and SEY6210 580  strain background (table S1). The FACS 581 screen for genes affecting the starvation-induced endocytosis of Mup1-pHluorin was performed using 582 the non-essential gene deletion strain collection purchased from Open Biosystems (BY4742: MATα 583 acquisition. GuavaSoft 2.7 software was used for data analysis. The positive/negative cut-off was set 632 for each plate empirically at the intercept of the log/starvation histograms of the WT and art2∆ controls 633 (art2∆, which emerged as a well-reproducible hit early in the screen, was included as a negative control 634 in all further plates). All potential hits were re-examined by fluorescence microscopy. To this end, at 635 least 100 starved cells were analyzed by fluorescence microscopy after starvation and the percentage of 636 cells showing a degradation-deficient phenotype (Mup1-pHluorin at the plasma membrane, in small 637 cytosolic objects, class E-like objects or small objects within vacuoles) of the total number of cells 638 counted was calculated (table S2). Strains with more than 45% cells with retained fluorescence after at 639 least 18 hours of starvation were considered as hits. For a stringent final selection, we compared those 640 hits to the original FACS screen and finally only considered those in which at least once more the 30% 641 Mup1-pHluorin fluorescence was also retained after starvation in the FACS screen. In addition, most 642 hits were also scored for methionine-induced endocytosis of Mup1-pHluorin. Hits were considered 643 starvation-specific if the fluorescence was quenched in more than 67% of cells after 90 minutes of 644 methionine treatment (20µg/ml). 645 646

Mass spectrometry sample preparation and analysis. 721
Coomassie-stained gel bands were excised from SDS-PAGE gels, reduced with dithiothreitol, alkylated 722 23 with iodoacetamide and digested with trypsin (Promega) as previously described (Faserl et al., 2019). 723 Tryptic digest were analyzed using an UltiMate 3000 RSCLnano-HPLC system coupled to a Q Exactive 724 HF mass spectrometer (both Thermo Scientific, Bremen, Germany) equipped with a Nanospray Flex 725 ionization source. The peptides were separated on a homemade fritless fused-silica micro-capillary 726 column (75 µm i.d. x 280 µm o.d. x 10 cm length) packed with 3.0 µm reversed-phase C18 material. 727 Solvents for HPLC were 0.1% formic acid (solvent A) and 0.1% formic acid in 85% acetonitrile (solvent 728 B). The gradient profile was as follows: 0-4 min, 4% B; 4-57 min, 4-35% B; 57-62 min, 35-100% B, 729 and 62-67 min, 100 % B. The flow rate was 250 nL/min. 730 The Q Exactive HF mass spectrometer was operating in the data dependent mode selecting the top 20 731 most abundant isotope patterns with charge >1 from the survey scan with an isolation window of 1.6 732 mass-to-charge ratio (m/z). Survey full scan MS spectra were acquired from 300 to 1750 m/z at a 733 resolution of 60,000 with a maximum injection time (IT) of 120 ms, and automatic gain control (AGC) 734 target 1e6. The selected isotope patterns were fragmented by higher-energy collisional dissociation with 735 normalized collision energy of 28 at a resolution of 30,000 with a maximum IT of 120 ms, and AGC 736 target 5e5. 737 Data Analysis was performed using Proteome Discoverer 4.1 (Thermo Scientific) with search engine 738 Sequest. The raw files were searched against yeast database (orf_trans_all) with sequence of Mup1-GFP 739 added. Precursor and fragment mass tolerance was set to 10 ppm and 0.02 Da, respectively, and up to 740 two missed cleavages were allowed. Carbamidomethylation of cysteine was set as static modification. 741 Oxidation of methionine, ubiquitination of lysine, and phosphorylation of serine threonine, and tyrosine 742 were set as variable modifications. Peptide identifications were filtered at 1% false discovery rate. 743

Figure 1: Amino acid and nitrogen starvation triggers broad but specific endocytosis and 1 lysosomal degradation of plasma membrane proteins. 2
A) Left: a library of 147 yeast strains expressing chromosomally GFP-tagged membrane proteins was 3 tested for plasma membrane (PM) localization during nutrient replete exponential growth. Right: 4 verified PM proteins were starved for amino acids and nitrogen (-N) 6-8h or treated with 20 µg/ml L-5 methionine (+Met) after 24h of exponential growth. The localization of GFP was assayed by 6 fluorescence microscopy. B) Summary of the phenotypes of GFP-tagged PM proteins during starvation. 7 Indicated are numbers of PM proteins that are down-regulated, up-regulated or unchanged compared to 8 the exponential growth phase, each exemplified by one representative strain. PM: plasma membrane; 9 V: vacuole. Scale bars = 5 µm. See also Fig. S1 and Table S1.     Live-cell fluorescence microscopy analysis of art2∆ cells expressing TAT2-GFP and pRS416-ART2, 206 empty vector or pRS416-ART2 K664D,R665D,R666D,K667D. Cells were starved (-N) for 6h after 24h 207 exponential growth. Scale bars = 5 µm. 208