FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W1987181427> ?p ?o ?g. }

• W1987181427 endingPage "260" @default.
• W1987181427 startingPage "249" @default.
• W1987181427 abstract "•Prion-like domains can be trans-Golgi network retention signals•PLDs can reversibly shift the equilibrium of protein distribution•PLDs may act as stress-response elements•Exomer-dependent cargoes need tightly regulated endo- and exocytosis for localization Prion and prion-like domains (PLDs) are found in many proteins throughout the animal kingdom. We found that the PLD in the S. cerevisiae exomer-dependent cargo protein Pin2 is involved in the regulation of protein transport and localization. The domain serves as a Pin2 retention signal in the trans-Golgi network (TGN). Pin2 is localized in a polarized fashion at the plasma membrane of the bud early in the cell cycle and the bud neck at cytokinesis. This polarized localization is dependent on both exo- and endocytosis. Upon environmental stress, Pin2 is rapidly endocytosed, and the PLD aggregates and causes sequestration of Pin2. The aggregation of Pin2 is reversible upon stress removal and Pin2 is rapidly re-exported to the plasma membrane. Altogether, these data uncover a role for PLDs as protein localization elements. Prion and prion-like domains (PLDs) are found in many proteins throughout the animal kingdom. We found that the PLD in the S. cerevisiae exomer-dependent cargo protein Pin2 is involved in the regulation of protein transport and localization. The domain serves as a Pin2 retention signal in the trans-Golgi network (TGN). Pin2 is localized in a polarized fashion at the plasma membrane of the bud early in the cell cycle and the bud neck at cytokinesis. This polarized localization is dependent on both exo- and endocytosis. Upon environmental stress, Pin2 is rapidly endocytosed, and the PLD aggregates and causes sequestration of Pin2. The aggregation of Pin2 is reversible upon stress removal and Pin2 is rapidly re-exported to the plasma membrane. Altogether, these data uncover a role for PLDs as protein localization elements. Prion proteins can exist in a normally folded state or an aggregated state. The aggregated state is able to drive normally folded proteins into aggregation. Induction of the yeast Sup35 prion [PSI+] can occur spontaneously, but is greatly facilitated if the cell has previously achieved a [PIN+] state (Derkatch et al., 1997Derkatch I.L. Bradley M.E. Zhou P. Chernoff Y.O. Liebman S.W. Genetic and environmental factors affecting the de novo appearance of the [PSI+] prion in Saccharomyces cerevisiae.Genetics. 1997; 147: 507-519Crossref PubMed Google Scholar). This [PIN+] state can be reached by the overexpression of a number of different factors that contain a prion domain or prion-like domain (PLD) (Derkatch et al., 2001Derkatch I.L. Bradley M.E. Hong J.Y. Liebman S.W. Prions affect the appearance of other prions: the story of [PIN(+)].Cell. 2001; 106: 171-182Abstract Full Text Full Text PDF PubMed Scopus (478) Google Scholar). Thus, efficient induction of prions may require the presence of other prions. Genome-wide analyses indicate that 0.3% (in humans) to 24% (in plasmodium) of cellular proteins contain a prion domain or PLD (Michelitsch and Weissman, 2000Michelitsch M.D. Weissman J.S. A census of glutamine/asparagine-rich regions: implications for their conserved function and the prediction of novel prions.Proc. Natl. Acad. Sci. USA. 2000; 97: 11910-11915Crossref PubMed Scopus (364) Google Scholar, Osherovich and Weissman, 2002Osherovich L.Z. Weissman J.S. The utility of prions.Dev. Cell. 2002; 2: 143-151Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar, Singh et al., 2004Singh G.P. Chandra B.R. Bhattacharya A. Akhouri R.R. Singh S.K. Sharma A. Hyper-expansion of asparagines correlates with an abundance of proteins with prion-like domains in Plasmodium falciparum.Mol. Biochem. Parasitol. 2004; 137: 307-319Crossref PubMed Scopus (72) Google Scholar). RNA-binding proteins are overrepresented among the PLD-containing proteins (Michelitsch and Weissman, 2000Michelitsch M.D. Weissman J.S. A census of glutamine/asparagine-rich regions: implications for their conserved function and the prediction of novel prions.Proc. Natl. Acad. Sci. USA. 2000; 97: 11910-11915Crossref PubMed Scopus (364) Google Scholar). Obviously, not all prion or PLDs cause disease, and they may instead act as scaffold or interaction domains. Yet, in most instances, their precise role remains elusive. Transport to the plasma membrane and secretion are essential processes in eukaryotic cells. Cargoes destined for the plasma membrane will be sorted into transport carriers for either direct delivery or delivery via endosomes. Evidence for the direct route exists in yeast and in mammalian cells. TGN46-containing transport containers, which are dependent on protein kinase D and are devoid of vesicular stomatitis virus glycoprotein (VSVG) or collagen, have been identified in HeLa cells, indicating a specific sorting mechanism at the trans-Golgi network (TGN) (Wakana et al., 2012Wakana Y. van Galen J. Meissner F. Scarpa M. Polishchuk R.S. Mann M. Malhotra V. A new class of carriers that transport selective cargo from the trans Golgi network to the cell surface.EMBO J. 2012; 31: 3976-3990Crossref PubMed Scopus (62) Google Scholar). In Saccharomyces cerevisiae, the chitin synthase Chs3 and the mating-response protein Fus1 require Chs5 and Chs5p-Arf1p-binding proteins (ChAPs) collectively termed exomer for their export from the TGN to the plasma membrane (Trautwein et al., 2006Trautwein M. Schindler C. Gauss R. Dengjel J. Hartmann E. Spang A. Arf1p, Chs5p and the ChAPs are required for export of specialized cargo from the Golgi.EMBO J. 2006; 25: 943-954Crossref PubMed Scopus (69) Google Scholar, Wang et al., 2006Wang C.W. Hamamoto S. Orci L. Schekman R. Exomer: A coat complex for transport of select membrane proteins from the trans-Golgi network to the plasma membrane in yeast.J. Cell Biol. 2006; 174: 973-983Crossref PubMed Scopus (94) Google Scholar). Chs3 and Fus1 necessitate a combination of regulated endocytosis and exocytosis to achieve their precise localization at the bud neck for Chs3 and to the bud tip for Fus1 in a cell-cycle-dependent manner (Barfield et al., 2009Barfield R.M. Fromme J.C. Schekman R. The exomer coat complex transports Fus1p to the plasma membrane via a novel plasma membrane sorting signal in yeast.Mol. Biol. Cell. 2009; 20: 4985-4996Crossref PubMed Scopus (48) Google Scholar, Valdivia et al., 2002Valdivia R.H. Baggott D. Chuang J.S. Schekman R.W. The yeast clathrin adaptor protein complex 1 is required for the efficient retention of a subset of late Golgi membrane proteins.Dev. Cell. 2002; 2: 283-294Abstract Full Text Full Text PDF PubMed Scopus (168) Google Scholar). The ChAP family consists of Bch1, Bch2, Bud7, and Chs6, which can associate with Chs5 to form oligomers of heterotetrameric complexes. These complexes consist of two Chs5 molecules and two ChAPs, and either two identical or two different ChAPs can be bound to Chs5 (Paczkowski et al., 2012Paczkowski J.E. Richardson B.C. Strassner A.M. Fromme J.C. The exomer cargo adaptor structure reveals a novel GTPase-binding domain.EMBO J. 2012; 31: 4191-4203Crossref PubMed Scopus (26) Google Scholar, Trautwein et al., 2006Trautwein M. Schindler C. Gauss R. Dengjel J. Hartmann E. Spang A. Arf1p, Chs5p and the ChAPs are required for export of specialized cargo from the Golgi.EMBO J. 2006; 25: 943-954Crossref PubMed Scopus (69) Google Scholar). The ChAPs may act as adaptor molecules to interact and recruit cargo to specific sites at the TGN from which they reach the plasma membrane. Although previous studies identified Chs3 and Fus1 motifs that were necessary for export from the TGN, none of these motifs were sufficient (Barfield et al., 2009Barfield R.M. Fromme J.C. Schekman R. The exomer coat complex transports Fus1p to the plasma membrane via a novel plasma membrane sorting signal in yeast.Mol. Biol. Cell. 2009; 20: 4985-4996Crossref PubMed Scopus (48) Google Scholar, Rockenbauch et al., 2012Rockenbauch U. Ritz A.M. Sacristan C. Roncero C. Spang A. The complex interactions of Chs5p, the ChAPs, and the cargo Chs3p.Mol. Biol. Cell. 2012; 23: 4402-4415Crossref PubMed Scopus (17) Google Scholar, Starr et al., 2012Starr T.L. Pagant S. Wang C.W. Schekman R. Sorting signals that mediate traffic of chitin synthase III between the TGN/endosomes and to the plasma membrane in yeast.PLoS ONE. 2012; 7: e46386Crossref PubMed Scopus (30) Google Scholar). In addition, the interaction motifs were not conserved between the two cargo proteins. Thus, the interaction between the cargoes and exomer appears to be rather complex. Given the lack of conserved motifs between Chs3 and Fus1, other interaction sites must be important for controlling the export of these proteins in a temporally and spatially controlled manner. These interaction sites potentially could adopt an appropriate conformation upon interaction with the ChAPs, and then the linear transport signal might be recognized. In support of this notion, it was shown that all ChAPs are able to interact with Chs3, although only Chs6 is essential for its plasma membrane localization (Trautwein et al., 2006Trautwein M. Schindler C. Gauss R. Dengjel J. Hartmann E. Spang A. Arf1p, Chs5p and the ChAPs are required for export of specialized cargo from the Golgi.EMBO J. 2006; 25: 943-954Crossref PubMed Scopus (69) Google Scholar). Examples of such interactions include Src homology domains that recognize phosphorylated Tyr in proteins (Groffen et al., 1983Groffen J. Heisterkamp N. Reynolds Jr., F.H. Stephenson J.R. Homology between phosphotyrosine acceptor site of human c-abl and viral oncogene products.Nature. 1983; 304: 167-169Crossref PubMed Scopus (48) Google Scholar, Moran et al., 1990Moran M.F. Koch C.A. Anderson D. Ellis C. England L. Martin G.S. Pawson T. Src homology region 2 domains direct protein-protein interactions in signal transduction.Proc. Natl. Acad. Sci. USA. 1990; 87: 8622-8626Crossref PubMed Scopus (326) Google Scholar), the interaction of the ArfGAP Glo3 with SNAREs and cargo (Rein et al., 2002Rein U. Andag U. Duden R. Schmitt H.D. Spang A. ARF-GAP-mediated interaction between the ER-Golgi v-SNAREs and the COPI coat.J. Cell Biol. 2002; 157: 395-404Crossref PubMed Scopus (96) Google Scholar, Schindler et al., 2009Schindler C. Rodriguez F. Poon P.P. Singer R.A. Johnston G.C. Spang A. The GAP domain and the SNARE, coatomer and cargo interaction region of the ArfGAP2/3 Glo3 are sufficient for Glo3 function.Traffic. 2009; 10: 1362-1375Crossref PubMed Scopus (25) Google Scholar), and PLD-containing proteins that are important for processing body and stress granule assembly (Alberti et al., 2009Alberti S. Halfmann R. King O. Kapila A. Lindquist S. A systematic survey identifies prions and illuminates sequence features of prionogenic proteins.Cell. 2009; 137: 146-158Abstract Full Text Full Text PDF PubMed Scopus (715) Google Scholar, Decker et al., 2007Decker C.J. Teixeira D. Parker R. Edc3p and a glutamine/asparagine-rich domain of Lsm4p function in processing body assembly in Saccharomyces cerevisiae.J. Cell Biol. 2007; 179: 437-449Crossref PubMed Scopus (347) Google Scholar, Gilks et al., 2004Gilks N. Kedersha N. Ayodele M. Shen L. Stoecklin G. Dember L.M. Anderson P. Stress granule assembly is mediated by prion-like aggregation of TIA-1.Mol. Biol. Cell. 2004; 15: 5383-5398Crossref PubMed Scopus (707) Google Scholar, Michelitsch and Weissman, 2000Michelitsch M.D. Weissman J.S. A census of glutamine/asparagine-rich regions: implications for their conserved function and the prediction of novel prions.Proc. Natl. Acad. Sci. USA. 2000; 97: 11910-11915Crossref PubMed Scopus (364) Google Scholar, Vessey et al., 2006Vessey J.P. Vaccani A. Xie Y. Dahm R. Karra D. Kiebler M.A. Macchi P. Dendritic localization of the translational repressor Pumilio 2 and its contribution to dendritic stress granules.J. Neurosci. 2006; 26: 6496-6508Crossref PubMed Scopus (143) Google Scholar) or are often found in cytoskeletal elements (Alberti et al., 2009Alberti S. Halfmann R. King O. Kapila A. Lindquist S. A systematic survey identifies prions and illuminates sequence features of prionogenic proteins.Cell. 2009; 137: 146-158Abstract Full Text Full Text PDF PubMed Scopus (715) Google Scholar, Michelitsch and Weissman, 2000Michelitsch M.D. Weissman J.S. A census of glutamine/asparagine-rich regions: implications for their conserved function and the prediction of novel prions.Proc. Natl. Acad. Sci. USA. 2000; 97: 11910-11915Crossref PubMed Scopus (364) Google Scholar). Chs3 is a multispanning transmembrane protein, and Fus1 only becomes exomer dependent upon mating. To better understand the exomer-dependent transport pathway, we identified cargo proteins, including Pin2, a single transmembrane domain (TMD) protein with a large cytoplasmic region that contains a PLD. This PLD regulates the traffic of Pin2 under normal growth conditions and is essential for Pin2 retention in internal structures upon stress. We identified a transport mechanism in which a PLD is essential for the temporal and spatial control of intracellular protein localization. In order to better understand exomer-dependent transport to the plasma membrane, we aimed to identify novel cargoes. We appended Chs5 or each of the four members of the ChAP family with a histidine-biotin-histidine (HBH) tag. The HBH tag consists of a biotinylation sequence flanked by two His6 tags (Tagwerker et al., 2006Tagwerker C. Zhang H. Wang X. Larsen L.S. Lathrop R.H. Hatfield G.W. Auer B. Huang L. Kaiser P. HB tag modules for PCR-based gene tagging and tandem affinity purification in Saccharomyces cerevisiae.Yeast. 2006; 23: 623-632Crossref PubMed Scopus (44) Google Scholar). This tag allows the purification of proteins or (after crosslinking) protein complexes under denaturing conditions. This tag should allow for easy extraction of membrane proteins, which would represent potential cargo proteins, when bound to the exomer complex. Cells were crosslinked and lysed under denaturing conditions. The crosslinked complexes were affinity purified and analyzed by mass spectrometry (Figure 1A). We identified TMD-containing proteins (potential cargoes) and soluble proteins (potential regulators). We focused on potential cargoes and tested them for their ability to be transported to the plasma membrane in a Chs5-dependent manner. One of the hits that required Chs5 for localization to the bud in small- and medium-sized cells and to the bud neck in large-budded cells was the previously uncharacterized PLD-containing protein Pin2 (Figure 1B). In the absence of Chs5, Pin2 remained in internal structures, similar to what was observed with the other exomer-dependent cargoes (Barfield et al., 2009Barfield R.M. Fromme J.C. Schekman R. The exomer coat complex transports Fus1p to the plasma membrane via a novel plasma membrane sorting signal in yeast.Mol. Biol. Cell. 2009; 20: 4985-4996Crossref PubMed Scopus (48) Google Scholar, Santos and Snyder, 1997Santos B. Snyder M. Targeting of chitin synthase 3 to polarized growth sites in yeast requires Chs5p and Myo2p.J. Cell Biol. 1997; 136: 95-110Crossref PubMed Scopus (169) Google Scholar, Trautwein et al., 2006Trautwein M. Schindler C. Gauss R. Dengjel J. Hartmann E. Spang A. Arf1p, Chs5p and the ChAPs are required for export of specialized cargo from the Golgi.EMBO J. 2006; 25: 943-954Crossref PubMed Scopus (69) Google Scholar). If Pin2 is an exomer-dependent cargo, a deletion of all four ChAPs should phenocopy a Δchs5 strain. In a Δ4ChAPs strain, Pin2 was also found in internal structures (Figure 1B). Therefore, Pin2 represents a exomer-dependent cargo. It has been reported that all exomer-dependent cargoes localize to the bud or bud neck (Barfield et al., 2009Barfield R.M. Fromme J.C. Schekman R. The exomer coat complex transports Fus1p to the plasma membrane via a novel plasma membrane sorting signal in yeast.Mol. Biol. Cell. 2009; 20: 4985-4996Crossref PubMed Scopus (48) Google Scholar, Chuang and Schekman, 1996Chuang J.S. Schekman R.W. Differential trafficking and timed localization of two chitin synthase proteins, Chs2p and Chs3p.J. Cell Biol. 1996; 135: 597-610Crossref PubMed Scopus (188) Google Scholar, Santos and Snyder, 1997Santos B. Snyder M. Targeting of chitin synthase 3 to polarized growth sites in yeast requires Chs5p and Myo2p.J. Cell Biol. 1997; 136: 95-110Crossref PubMed Scopus (169) Google Scholar), suggesting that perhaps all bud-localized proteins are potential exomer clients. However, another candidate from our biochemical screen, Skg6, which localized to the bud and the bud neck in a cell-cycle-dependent manner similar to that observed for Pin2, reached the plasma membrane through an exomer-independent pathway, because deletion of CHS5 or the four ChAPs had no effect on Skg6 localization (Figure 1B). Thus, the cell-cycle-dependent spatial distribution of the proteins alone cannot be used to discriminate between exomer-dependent and -independent cargoes. Pin2 and Skg6 are single TMD proteins with unclear topology. To determine the topology of both proteins, we performed trypsin digests of cells expressing chromosomal C-terminal GFP fusions of Pin2 and Skg6 in the presence or absence of 1% TX-100. Pin2-GFP was resistant to trypsin treatment for up to 90 min in the absence of detergent. Solubilizing the plasma membrane rendered Pin2-GFP protease sensitive (Figure 1C). Similar results were obtained for Skg6-GFP. Consistent with these results, phosphoproteome studies reported phosphorylation sites for both Skg6 and Pin2 in the C-terminal part of the proteins (Bodenmiller et al., 2007Bodenmiller B. Malmstrom J. Gerrits B. Campbell D. Lam H. Schmidt A. Rinner O. Mueller L.N. Shannon P.T. Pedrioli P.G. et al.PhosphoPep—a phosphoproteome resource for systems biology research in Drosophila Kc167 cells.Mol. Syst. Biol. 2007; 3: 139Crossref PubMed Scopus (156) Google Scholar, Li et al., 2007Li X. Gerber S.A. Rudner A.D. Beausoleil S.A. Haas W. Villén J. Elias J.E. Gygi S.P. Large-scale phosphorylation analysis of alpha-factor-arrested Saccharomyces cerevisiae.J. Proteome Res. 2007; 6: 1190-1197Crossref PubMed Scopus (258) Google Scholar, Sadowski et al., 2013Sadowski I. Breitkreutz B.J. Stark C. Su T.C. Dahabieh M. Raithatha S. Bernhard W. Oughtred R. Dolinski K. Barreto K. Tyers M. The PhosphoGRID Saccharomyces cerevisiae protein phosphorylation site database: version 2.0 update.Database (Oxford). 2013; 2013: bat026Crossref PubMed Scopus (77) Google Scholar, Soulard et al., 2010Soulard A. Cremonesi A. Moes S. Schütz F. Jenö P. Hall M.N. The rapamycin-sensitive phosphoproteome reveals that TOR controls protein kinase A toward some but not all substrates.Mol. Biol. Cell. 2010; 21: 3475-3486Crossref PubMed Scopus (182) Google Scholar). Therefore, the C terminus of Pin2 and Skg6 face the cytoplasm, and the N terminus of either protein is exposed to the environment. In both proteins, the TMD is relatively close to the N terminus, resulting in small extracellular domains (Figure 1D). The determination of the topology allowed us to create GST-fusion proteins of the cytoplasmic exposed tails of Pin2 and Skg6, and to revisit their interaction with exomer. We wanted to confirm the interaction because crosslinking only measures proximity. We performed a GST pull-down experiment from yeast lysates in which three of the four ChAPs (Bch1, Bch2, and Bud7) were chromosomally appended with different tags, or Chs6 was myc tagged. The functionality of the tagged proteins was established previously (Trautwein et al., 2006Trautwein M. Schindler C. Gauss R. Dengjel J. Hartmann E. Spang A. Arf1p, Chs5p and the ChAPs are required for export of specialized cargo from the Golgi.EMBO J. 2006; 25: 943-954Crossref PubMed Scopus (69) Google Scholar). Pin2 and Skg6, but not the endoplasmic reticulum (ER)-Golgi v-SNARE Sec22, pulled down exomer components (Figure 1E). Interestingly, proximity to and even interaction with exomer appears to be insufficient to describe an exomer-dependent cargo, as Skg6 travels to the plasma membrane in the absence of exomer. It is conceivable, however, that Skg6 can use either pathway to reach the plasma membrane. As a final proof, we probed the interaction of Pin2 with Chs5. Chs3 and Fus1 depend on the ChAPs for efficient interaction with Chs5 (Rockenbauch et al., 2012Rockenbauch U. Ritz A.M. Sacristan C. Roncero C. Spang A. The complex interactions of Chs5p, the ChAPs, and the cargo Chs3p.Mol. Biol. Cell. 2012; 23: 4402-4415Crossref PubMed Scopus (17) Google Scholar, Sanchatjate and Schekman, 2006Sanchatjate S. Schekman R. Chs5/6 complex: a multiprotein complex that interacts with and conveys chitin synthase III from the trans-Golgi network to the cell surface.Mol. Biol. Cell. 2006; 17: 4157-4166Crossref PubMed Scopus (59) Google Scholar). Although we could detect a robust interaction between Chs5 and Pin2 in the presence of the ChAPs, this interaction was abolished when the ChAPs were deleted (Figure 1E). Therefore, we conclude that Pin2 is a exomer-dependent cargo. Exomer-dependent cargoes require one or two members of the ChAP family for timely exit from the TGN (Barfield et al., 2009Barfield R.M. Fromme J.C. Schekman R. The exomer coat complex transports Fus1p to the plasma membrane via a novel plasma membrane sorting signal in yeast.Mol. Biol. Cell. 2009; 20: 4985-4996Crossref PubMed Scopus (48) Google Scholar, Sanchatjate and Schekman, 2006Sanchatjate S. Schekman R. Chs5/6 complex: a multiprotein complex that interacts with and conveys chitin synthase III from the trans-Golgi network to the cell surface.Mol. Biol. Cell. 2006; 17: 4157-4166Crossref PubMed Scopus (59) Google Scholar, Trautwein et al., 2006Trautwein M. Schindler C. Gauss R. Dengjel J. Hartmann E. Spang A. Arf1p, Chs5p and the ChAPs are required for export of specialized cargo from the Golgi.EMBO J. 2006; 25: 943-954Crossref PubMed Scopus (69) Google Scholar, Ziman et al., 1998Ziman M. Chuang J.S. Tsung M. Hamamoto S. Schekman R. Chs6p-dependent anterograde transport of Chs3p from the chitosome to the plasma membrane in Saccharomyces cerevisiae.Mol. Biol. Cell. 1998; 9: 1565-1576Crossref PubMed Scopus (120) Google Scholar). We tested single and double ChAP deletions for their failure to export Pin2-GFP from the TGN. Only the double deletion Δbch1 Δbch2 altered Pin2-GFP localization (Figures S1A–S1C). However, this mutant was less potent than Δchs5, indicating that the other ChAPs also can contribute to proper Pin2 localization (Figure S1C). Next, we asked whether either Bch1 or Bch2 would also be sufficient for Pin2 TGN export. Pin2 still reached the plasma membrane even when only one ChAP was present, albeit somewhat less efficiently than in the wild-type (WT; Figures S1D and S1E). At least in the case of Bch2, this small reduction in export efficiency was not due to less binding to Pin2 (Figure S1F). Our data indicate that Bch1 and Bch2 can independently promote export of Pin2 from the TGN to the plasma membrane. Thus far, we have shown that Pin2 is an exomer-dependent cargo that binds directly to exomer. The interaction of exomer with its cargoes Chs3 and Fus1 is complex and requires more than just a linear sequence motif (Barfield et al., 2009Barfield R.M. Fromme J.C. Schekman R. The exomer coat complex transports Fus1p to the plasma membrane via a novel plasma membrane sorting signal in yeast.Mol. Biol. Cell. 2009; 20: 4985-4996Crossref PubMed Scopus (48) Google Scholar, Rockenbauch et al., 2012Rockenbauch U. Ritz A.M. Sacristan C. Roncero C. Spang A. The complex interactions of Chs5p, the ChAPs, and the cargo Chs3p.Mol. Biol. Cell. 2012; 23: 4402-4415Crossref PubMed Scopus (17) Google Scholar). To identify potential interacting regions, we generated three truncations in the cytoplasmic domain containing a GST-Pin2 construct. As shown above, the cytoplasmic domain of Pin2 bound all ChAP proteins (Figures 1E and 2A ). In addition, the construct expressing the C-terminal ∼120 amino acids of Pin2 precipitated the ChAPs, albeit more weakly. Since GST-Pin2(72-210) was unable to interact with exomer, we conclude that the exomer-binding site resides in the C-terminal 72 amino acids. Thus, exomer recognizes sequences in the Pin2 C terminus, but other sequences in the molecule might still contribute to the binding efficacy. Next we wanted to test whether the C-terminal part of Pin2 is necessary and sufficient to cause exomer-dependent export (Figure 2B). First, we generated a construct in which the exomer interaction site identified in vitro was eliminated (Pin2(1-210)-GFP). This construct still reached the plasma membrane in the WT and to a lesser extent in Δchs5; however, the polarized localization was lost (Figures 2B and 2C). This phenotype is reminiscent of Chs3 localization in Δchs5 Δapm1 cells, in which recycling from endosomes to the TGN is blocked (Valdivia et al., 2002Valdivia R.H. Baggott D. Chuang J.S. Schekman R.W. The yeast clathrin adaptor protein complex 1 is required for the efficient retention of a subset of late Golgi membrane proteins.Dev. Cell. 2002; 2: 283-294Abstract Full Text Full Text PDF PubMed Scopus (168) Google Scholar). A construct that contained only the first 245 of the 282 amino acids of Pin2 still accumulated at the plasma membrane in an exomer-dependent manner. Therefore, the exomer interaction site might reside in residues 210–245 of Pin2. Trimming the protein further down to 152 residues shifted Pin2 localization entirely to the plasma membrane and the internal pool was depleted, consistent with a defect in endocytosis (Figure 2B). A similar phenotype was previusly reported for Chs3 localization in a Δend3 strain, in which endocytosis was blocked (Chuang and Schekman, 1996Chuang J.S. Schekman R.W. Differential trafficking and timed localization of two chitin synthase proteins, Chs2p and Chs3p.J. Cell Biol. 1996; 135: 597-610Crossref PubMed Scopus (188) Google Scholar, Ziman et al., 1996Ziman M. Chuang J.S. Schekman R.W. Chs1p and Chs3p, two proteins involved in chitin synthesis, populate a compartment of the Saccharomyces cerevisiae endocytic pathway.Mol. Biol. Cell. 1996; 7: 1909-1919Crossref PubMed Scopus (118) Google Scholar). Despite a notable plasma membrane localization of Pin2(Δ79-152)-GFP, most of the protein accumulated in the vacuole in WT cells, indicating that the membrane proximal region of Pin2 also may contribute to proper Pin2 localization. Most importantly, the pool that reached the plasma membrane arrived there in an exomer-independent manner, because localization of Pin2(Δ79-152)-GFP in Δchs5 was indistinguishable from that in WT cells. Therefore, the C-terminal domain is not sufficient to direct Pin2 into the exomer pathway. The effects we observed were not due to large overexpression of the constructs over the endogenous protein (Figure S2A). Taken together, these data indicate that the interaction between Pin2 and exomer might be rather complex, and it is rather unlikely that a short linear sequence within Pin2 would be necessary and sufficient to promote temporal and spatial controlled plasma membrane localization. These data are in agreement with what has been observed for the other exomer cargoes, Chs3 and Fus1 (Barfield et al., 2009Barfield R.M. Fromme J.C. Schekman R. The exomer coat complex transports Fus1p to the plasma membrane via a novel plasma membrane sorting signal in yeast.Mol. Biol. Cell. 2009; 20: 4985-4996Crossref PubMed Scopus (48) Google Scholar, Rockenbauch et al., 2012Rockenbauch U. Ritz A.M. Sacristan C. Roncero C. Spang A. The complex interactions of Chs5p, the ChAPs, and the cargo Chs3p.Mol. Biol. Cell. 2012; 23: 4402-4415Crossref PubMed Scopus (17) Google Scholar). Chs3 and Fus1 have been shown to reach endosomes and to be retrieved from there through an AP-1-dependent pathway (Barfield et al., 2009Barfield R.M. Fromme J.C. Schekman R. The exomer coat complex transports Fus1p to the plasma membrane via a novel plasma membrane sorting signal in yeast.Mol. Biol. Cell. 2009; 20: 4985-4996Crossref PubMed Scopus (48) Google Scholar, Valdivia et al., 2002Valdivia R.H. Baggott D. Chuang J.S. Schekman R.W. The yeast clathrin adaptor protein complex 1 is required for the efficient retention of a subset of late Golgi membrane proteins.Dev. Cell. 2002; 2: 283-294Abstract Full Text Full Text PDF PubMed Scopus (168) Google Scholar). In the absence of Chs5 and AP-1, Chs3 and Fus1 arrived at the plasma membrane through an alternative route. We tested whether this recycling is a general feature of exomer-dependent cargoes. As shown above, Pin2(1-210), but not Pin2(1-245), was localized to the plasma membrane independently of Chs5, indicating that the region aa 210–245 may contain an AP-1-binding site (Figure 2B). The μ subunits of AP complexes can bind to the Y-based sorting motif YXXø (where X is any amino acid, and ø is a bulky hydrophobic amino acid) (Ohno et al., 1995Ohno H. Stewart J. Fournier M.C. Bosshart H. Rhee I. Miyatake S. Saito T. Gallusser A. Kirchhausen T. Bonifacino J.S. Interaction of tyrosine-based sorting signals with clathrin-associated proteins.Science. 1995; 269: 1872-1875Crossref PubMed Scopus (814) Google Scholar). We identified a cryptic Y-based motif, YGENYYY, in the 210–245 peptide (Figure 3A). Although the spacing for the motif was not perfect, we replaced the Ys and N by As. Transport to the plasma membrane of the A mutant was independent of Chs5 (Figures 3B and 3C), indicating that YGENYYY could be a functional adaptor-complex-binding site. To prove that Pin2 indeed undergoes AP-1-dependent recycling, we deleted the μ subunit of the AP-1 complex, APM1. In a Δchs5 Δapm1 mutant, Pin2 was localized mostly at the plasma membrane, whereas Skg6 localization was not affected (Figures 3D and 3E). Moreover, the Pin2(AGEAAAA) mutant protein did not change its localization in a Δchs5 2Δapm1 mutant, confirming that YGENYYY" @default.
• W1987181427 created "2016-06-24" @default.
• W1987181427 creator A5005697687 @default.
• W1987181427 creator A5018813375 @default.
• W1987181427 creator A5038099252 @default.
• W1987181427 creator A5084319558 @default.
• W1987181427 date "2014-04-01" @default.
• W1987181427 modified "2023-10-16" @default.
• W1987181427 title "The Prion-like Domain in the Exomer-Dependent Cargo Pin2 Serves as a trans-Golgi Retention Motif" @default.
• W1987181427 cites W1519136326 @default.
• W1987181427 cites W1812300167 @default.
• W1987181427 cites W1894314352 @default.
• W1987181427 cites W1977677374 @default.
• W1987181427 cites W1979815209 @default.
• W1987181427 cites W1984122954 @default.
• W1987181427 cites W1984569087 @default.
• W1987181427 cites W1989390455 @default.
• W1987181427 cites W1994528127 @default.
• W1987181427 cites W2006124679 @default.
• W1987181427 cites W2010373663 @default.
• W1987181427 cites W2012385113 @default.
• W1987181427 cites W2012920767 @default.
• W1987181427 cites W2013453206 @default.
• W1987181427 cites W2016334729 @default.
• W1987181427 cites W2035672439 @default.
• W1987181427 cites W2040792888 @default.
• W1987181427 cites W2043676526 @default.
• W1987181427 cites W2044779763 @default.
• W1987181427 cites W2047091642 @default.
• W1987181427 cites W2063423234 @default.
• W1987181427 cites W2065436346 @default.
• W1987181427 cites W2089375674 @default.
• W1987181427 cites W2093213954 @default.
• W1987181427 cites W2100329713 @default.
• W1987181427 cites W2101201763 @default.
• W1987181427 cites W2106446195 @default.
• W1987181427 cites W2108398910 @default.
• W1987181427 cites W2108693323 @default.
• W1987181427 cites W2112488154 @default.
• W1987181427 cites W2119651404 @default.
• W1987181427 cites W2119918356 @default.
• W1987181427 cites W2120638418 @default.
• W1987181427 cites W2121189749 @default.
• W1987181427 cites W2125025556 @default.
• W1987181427 cites W2131286471 @default.
• W1987181427 cites W2132121416 @default.
• W1987181427 cites W2136176984 @default.
• W1987181427 cites W2138906088 @default.
• W1987181427 cites W2146865209 @default.
• W1987181427 cites W2148497285 @default.
• W1987181427 cites W2149541702 @default.
• W1987181427 cites W2153618281 @default.
• W1987181427 cites W2158714857 @default.
• W1987181427 cites W2158748930 @default.
• W1987181427 cites W2161651587 @default.
• W1987181427 cites W2162930176 @default.
• W1987181427 cites W2170283197 @default.
• W1987181427 cites W2323539313 @default.
• W1987181427 doi "https://doi.org/10.1016/j.celrep.2014.02.026" @default.
• W1987181427 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/24656818" @default.
• W1987181427 hasPublicationYear "2014" @default.
• W1987181427 type Work @default.
• W1987181427 sameAs 1987181427 @default.
• W1987181427 citedByCount "24" @default.
• W1987181427 countsByYear W19871814272014 @default.
• W1987181427 countsByYear W19871814272015 @default.
• W1987181427 countsByYear W19871814272016 @default.
• W1987181427 countsByYear W19871814272017 @default.
• W1987181427 countsByYear W19871814272018 @default.
• W1987181427 countsByYear W19871814272020 @default.
• W1987181427 countsByYear W19871814272021 @default.
• W1987181427 countsByYear W19871814272022 @default.
• W1987181427 countsByYear W19871814272023 @default.
• W1987181427 crossrefType "journal-article" @default.
• W1987181427 hasAuthorship W1987181427A5005697687 @default.
• W1987181427 hasAuthorship W1987181427A5018813375 @default.
• W1987181427 hasAuthorship W1987181427A5038099252 @default.
• W1987181427 hasAuthorship W1987181427A5084319558 @default.
• W1987181427 hasBestOaLocation W19871814271 @default.
• W1987181427 hasConcept C119062480 @default.
• W1987181427 hasConcept C121332964 @default.
• W1987181427 hasConcept C158617107 @default.
• W1987181427 hasConcept C185592680 @default.
• W1987181427 hasConcept C24890656 @default.
• W1987181427 hasConcept C32276052 @default.
• W1987181427 hasConcept C86803240 @default.
• W1987181427 hasConcept C95444343 @default.
• W1987181427 hasConceptScore W1987181427C119062480 @default.
• W1987181427 hasConceptScore W1987181427C121332964 @default.
• W1987181427 hasConceptScore W1987181427C158617107 @default.
• W1987181427 hasConceptScore W1987181427C185592680 @default.
• W1987181427 hasConceptScore W1987181427C24890656 @default.
• W1987181427 hasConceptScore W1987181427C32276052 @default.
• W1987181427 hasConceptScore W1987181427C86803240 @default.
• W1987181427 hasConceptScore W1987181427C95444343 @default.
• W1987181427 hasIssue "1" @default.
• W1987181427 hasLocation W19871814271 @default.
• W1987181427 hasLocation W19871814272 @default.

• next