A PACS-1, GGA3 and CK2 complex regulates CI-MPR trafficking

The cation-independent mannose-6-phosphate receptor (CI-MPR) follows a highly regulated sorting itinerary to deliver hydrolases from the trans-Golgi network (TGN) to lysosomes. Cycling of CI-MPR between the TGN and early endosomes is mediated by GGA3, which directs TGN export, and PACS-1, which directs endosome-to-TGN retrieval. Despite executing opposing sorting steps, GGA3 and PACS-1 bind to an overlapping CI-MPR trafficking motif and their sorting activity is controlled by the CK2 phosphorylation of their respective autoregulatory domains. However, how CK2 coordinates these opposing roles is unknown. We report a CK2-activated phosphorylation cascade controlling PACS-1- and GGA3-mediated CI-MPR sorting. PACS-1 links GGA3 to CK2, forming a multimeric complex required for CI-MPR sorting. PACS-1-bound CK2 stimulates GGA3 phosphorylation, releasing GGA3 from CI-MPR and early endosomes. Bound CK2 also phosphorylates PACS-1Ser(278), promoting binding of PACS-1 to CI-MPR to retrieve the receptor to the TGN. Our results identify a CK2-controlled cascade regulating hydrolase trafficking and sorting of itinerant proteins in the TGN/endosomal system.     

Role of PACS-1 in trafficking of human cytomegalovirus glycoprotein B and virus production

The final envelopment of herpesviruses during assembly of new virions is thought to occur by the budding of core viral particles into a late secretory pathway organelle, the trans-Golgi network (TGN), or an associated endosomal compartment. Several herpesvirus envelope glycoproteins have been previously shown to localize to the TGN when expressed independently from other viral proteins. In at least some cases this TGN localization has been shown to be dependent on clusters of acidic residues within their cytoplasmic domains. Similar acidic cluster motifs are found in endogenous membrane proteins that also localize to the TGN. These acidic cluster motifs interact with PACS-1, a connector protein that is required for the trafficking of proteins containing such motifs from endosomes to the TGN. We show here that PACS-1 interacts with the cytoplasmic domain of the HCMV envelope glycoprotein B (gB) and that PACS-1 function is required for normal TGN localization of HCMV gB. Furthermore, inhibition of PACS-1 activity in infected cells leads to a decrease in HCMV titer, whereas an increase in expression of functional PACS-1 leads to an increase in HCMV titer, suggesting that PACS-1 is required for efficient production of HCMV.     

PACS-1 binding to adaptors is required for acidic cluster motif-mediated protein traffic

PACS-1 is a cytosolic protein involved in controlling the correct subcellular localization of integral membrane proteins that contain acidic cluster sorting motifs, such as furin and human immunodeficiency virus type 1 (HIV-1) NEF: We have now investigated the interaction of PACS-1 with heterotetrameric adaptor complexes. PACS-1 associates with both AP-1 and AP-3, but not AP-2, and forms a ternary complex between furin and AP-1. A short sequence within PACS-1 that is essential for binding to AP-1 has been identified. Mutation of this motif yielded a dominant-negative PACS-1 molecule that can still bind to acidic cluster motifs on cargo proteins but not to adaptor complexes. Expression of dominant-negative PACS-1 causes a mislocalization of both furin and mannose 6-phosphate receptor from the trans-Golgi network, but has no effect on the localization of proteins that do not contain acidic cluster sorting motifs. Furthermore, expression of dominant-negative PACS-1 inhibits the ability of HIV-1 Nef to downregulate MHC-I. These studies demonstrate the requirement for PACS-1 interactions with adaptor proteins in multiple processes, including secretory granule biogenesis and HIV-1 pathogenesis.     

An interdomain binding site on HIV-1 Nef interacts with PACS-1 and PACS-2 on endosomes to down-regulate MHC-I

The human immunodeficiency virus type 1 (HIV-1) accessory protein Nef directs virus escape from immune surveillance by subverting host cell intracellular signaling and membrane traffic to down-regulate cell-surface major histocompatibility complex class I (MHC-I). The interaction of Nef with the sorting proteins PACS-1 and PACS-2 mediates key signaling and trafficking steps required for Nef-mediated MHC-I down-regulation. Little is known, however, about the molecular basis underlying the Nef-PACS interaction. Here we identify the sites on Nef and the PACS proteins required for their interaction and describe the consequences of disrupting this interaction for Nef action. A previously unidentified cargo subsite on PACS-1 and PACS-2 interacted with a bipartite site on Nef formed by the EEEE(65) acidic cluster on the N-terminal domain and W(113) in the core domain. Mutation of these sites prevented the interaction between Nef and the PACS proteins on Rab5 (PACS-2 and PACS-1)- or Rab7 (PACS-1)-positive endosomes as determined by bimolecular fluorescence complementation and caused a Nef mutant defective in PACS binding to localize to distorted endosomal compartments. Consequently, disruption of the Nef-PACS interaction repressed Nef-induced MHC-I down-regulation in peripheral blood mononuclear cells. Our results provide insight into the molecular basis of Nef action and suggest new strategies to combat HIV-1.     

HIV-1 Nef binds PACS-2 to assemble a multikinase cascade that triggers major histocompatibility complex class I (MHC-I) down-regulation: analysis using short interfering RNA and knock-out mice

Human immunodeficiency virus, type 1, negative factor (Nef) initiates down-regulation of cell-surface major histocompatibility complex-I (MHC-I) by assembling an Src family kinase (SFK)-ZAP70/Syk-phosphoinositide 3-kinase (PI3K) cascade through the sequential actions of two sites, Nef EEEE(65) and PXXP(75). The internalized MHC-I molecules are then sequestered in endosomal compartments by a process requiring Nef Met(20). How Nef assembles the multikinase cascade to trigger the MHC-I down-regulation pathway is unknown. Here we report that EEEE(65)-dependent binding to the sorting protein PACS-2 targets Nef to the paranuclear region, enabling PXXP(75) to bind and activate a trans-Golgi network (TGN)-localized SFK. This SFK then phosphorylates ZAP-70 to recruit class I PI3K by interaction with the p85 C-terminal Src homology 2 domain. Using splenocytes and embryonic fibroblasts from PACS-2(-/-) mice, we confirm genetically that Nef requires PACS-2 to localize to the paranuclear region and assemble the multikinase cascade. Moreover, genetic loss of PACS-2 or inhibition of class I PI3K prevents Nef-mediated MHC-I down-regulation, demonstrating that short interfering RNA knockdown of PACS-2 phenocopies the gene knock-out. This PACS-2-dependent targeting pathway is not restricted to Nef, because PACS-2 is also required for trafficking of an endocytosed cation-independent mannose 6-phosphate receptor reporter from early endosomes to the TGN. Together, these results demonstrate PACS-2 is required for Nef action and sorting of itinerant membrane cargo in the TGN/endosomal system.     

HIV-1 Nef downregulates MHC-I by a PACS-1- and PI3K-regulated ARF6 endocytic pathway

The HIV-1 Nef-mediated downregulation of cell surface MHC-I molecules to the trans-Golgi network (TGN) enables HIV-1 to escape immune surveillance. However, the cellular pathway used by Nef to downregulate MHC-I is unknown. Here, we show that Nef and PACS-1 combine to usurp the ARF6 endocytic pathway by a PI3K-dependent process and downregulate cell surface MHC-I to the TGN. This mechanism requires the hierarchical actions of three Nef motifs-the acidic cluster 62EEEE(65), the SH3 domain binding site 72PXXP(75), and M(20)-in controlling PACS-1-dependent sorting to the TGN, ARF6 activation, and sequestering internalized MHC-I to the TGN, respectively. These data provide new insights into the cellular basis of HIV-1 immunoevasion.     

HIV-1 Nef protein binds to the cellular protein PACS-1 to downregulate class I major histocompatibility complexes

Major-histocompatibility-complex (MHC) proteins are used to display, on the surface of a cell, peptides derived from foreign material - such as a virus - that is infecting that cell. Cytotoxic T lymphocytes then recognize and kill the infected cell. The HIV-1 Nef protein downregulates the cell-surface expression of class I MHC proteins, and probably thereby promotes immune evasion by HIV-1. In the presence of Nef, class I MHC molecules are relocalized from the cell surface to the trans-Golgi network (TGN) through as-yet-unknown mechanisms. Here we show that Nef-induced downregulation of MHC-I expression and MHC-I targeting to the TGN require the binding of Nef to PACS-1, a molecule that controls the TGN localization of the cellular protein furin. This interaction is dependent on Nef's cluster of acidic amino acids. A chimaeric integral membrane protein containing Nef as its cytoplasmic domain localizes to the TGN after internalization, in an acidic-cluster- and PACS-1-dependent manner. These results support a model in which Nef relocalizes MHC-I by acting as a connector between MHC-I's cytoplasmic tail and the PACS-1-dependent protein-sorting pathway.     

The PC6B cytoplasmic domain contains two acidic clusters that direct sorting to distinct trans-Golgi network/endosomal compartments

The mammalian proprotein convertases (PCs) are a family of secretory pathway enzymes that catalyze the endoproteolytic maturation of peptide hormones and many bioactive proteins. Two PCs, furin and PC6B, are broadly expressed and share very similar cleavage site specificities, suggesting that they may be functionally redundant. However, germline knockout studies show that they are not. Here we report the distinct subcellular localization of PC6B and identify the sorting information within its cytoplasmic domain (cd). We show that in neuroendocrine cells, PC6B is localized to a paranuclear, brefeldin A-dispersible, BaCl(2)-responsive post-Golgi network (TGN) compartment distinct from furin and TGN38. The 88-amino acid PC6B-cd contains sorting information sufficient to direct reporter proteins to the same compartment as full-length PC6B. Mutational analysis indicates that endocytosis is predominantly directed by a canonical tyrosine-based motif (Tyr(1802)GluLysLeu). Truncation and sufficiency studies reveal that two clusters of acidic amino acids (ACs) within the PC6B-cd contain differential sorting information. The membrane-proximal AC (AC1) directs TGN localization and interacts with the TGN sorting protein PACS-1. The membrane-distal AC (AC2) promotes a localization characteristic of the full-length PC6B-cd. Our results demonstrate that AC motifs can target proteins to distinct TGN/endosomal compartments and indicate that the AC-mediated localization of PC6B and furin contribute to their distinct roles in vivo.     

Golgi-localizing, gamma-adaptin ear homology domain, ADP-ribosylation factor-binding (GGA) proteins interact with acidic dileucine sequences within the cytoplasmic domains of sorting receptors through their Vps27p/Hrs/STAM (VHS) domains

GGA (Golgi-localizing, gamma-adaptin ear homology domain, ARF-binding) proteins are potential effectors of ADP-ribosylation factors, are associated with the trans-Golgi network (TGN), and are involved in protein transport from this compartment. By yeast two-hybrid screening and subsequent two-hybrid and pull-down analyses, we have shown that GGA proteins, through their VHS (Vps27p/Hrs/STAM) domains, interact with acidic dileucine sequences found in the cytoplasmic domains of TGN-localized sorting receptors such as sortilin and mannose 6-phosphate receptor. A mutational analysis has revealed that a leucine pair and a cluster of acidic residues adjacent to the pair are mainly responsible for the interaction. A chimeric receptor with the sortilin cytoplasmic domain localizes to the TGN, whereas the chimeric receptor with a mutation at the leucine pair or the acidic cluster is mislocalized to punctate structures reminiscent of early endosomes. These results indicate that GGA proteins regulate the localization to or exit from the TGN of the sorting receptors.     

Phosphorylation by casein kinase 2 induces PACS-1 binding of nephrocystin and targeting to cilia

Mutations in proteins localized to cilia and basal bodies have been implicated in a growing number of human diseases. Access of these proteins to the ciliary compartment requires targeting to the base of the cilia. However, the mechanisms involved in transport of cilia proteins to this transitional zone are elusive. Here we show that nephrocystin, a ciliary protein mutated in the most prevalent form of cystic kidney disease in childhood, is expressed in respiratory epithelial cells and accumulates at the base of cilia, overlapping with markers of the basal body area and the transition zone. Nephrocystin interacts with the phosphofurin acidic cluster sorting protein (PACS)-1. Casein kinase 2 (CK2)-mediated phosphorylation of three critical serine residues within a cluster of acidic amino acids in nephrocystin mediates PACS-1 binding, and is essential for colocalization of nephrocystin with PACS-1 at the base of cilia. Inhibition of CK2 activity abrogates this interaction and results in the loss of correct nephrocystin targeting. These data suggest that CK2-dependent transport processes represent a novel pathway of targeting proteins to the cilia.     

The subcellular distribution of calnexin is mediated by PACS-2

Calnexin is an endoplasmic reticulum (ER) lectin that mediates protein folding on the rough ER. Calnexin also interacts with ER calcium pumps that localize to the mitochondria-associated membrane (MAM). Depending on ER homeostasis, varying amounts of calnexin target to the plasma membrane. However, no regulated sorting mechanism is so far known for calnexin. Our results now describe how the interaction of calnexin with the cytosolic sorting protein PACS-2 distributes calnexin between the rough ER, the MAM, and the plasma membrane. Under control conditions, more than 80% of calnexin localizes to the ER, with the majority on the MAM. PACS-2 knockdown disrupts the calnexin distribution within the ER and increases its levels on the cell surface. Phosphorylation by protein kinase CK2 of two calnexin cytosolic serines (Ser554/564) reduces calnexin binding to PACS-2. Consistent with this, a Ser554/564 Asp phosphomimic mutation partially reproduces PACS-2 knockdown by increasing the calnexin signal on the cell surface and reducing it on the MAM. PACS-2 knockdown does not reduce retention of other ER markers. Therefore, our results suggest that the phosphorylation state of the calnexin cytosolic domain and its interaction with PACS-2 sort this chaperone between domains of the ER and the plasma membrane.     

AP-1 recruitment to VAMP4 is modulated by phosphorylation-dependent binding of PACS-1

The R-SNARE VAMP4, which contains a dileucine motif, binds to the AP-1 (adaptor protein-1) subunit μ1a, but not μ1b, or the GGAs (Golgi-associated gamma ear containing ARF binding proteins). Serine 20 and leucines 25,26 are essential for this binding. AP-1 association with VAMP4 is enhanced when serine 30, in an acidic cluster, is phosphorylated by casein kinase 2. This phosphorylation-dependent modulation of AP-1 binding is mediated by PACS-1 (phosphofurin acidic cluster sorting protein). Ablation of both the dileucine motif and serine 30 results in a dramatic mislocalization of VAMP4 in the regulated secretory pathway in AtT20 cells. A dominant-negative PACS-1, which binds acidic clusters but not AP-1, also causes mislocalization of VAMP4. Our data support a model whereby phosphorylation-dependent recruitment of PACS-1 enhances AP-1 association to cargo, and suggest that efficient retrieval depends on the formation of a complex between cargo, such as VAMP4, AP-1 and PACS-1.     

HIV-1 Nef-induced down-regulation of MHC class I requires AP-1 and clathrin but not PACS-1 and is impeded by AP-2

Major histocompatibility complex class I is down-regulated from the surface of human immunodeficiency virus (HIV)-1-infected cells by Nef, a virally encoded protein that is thought to reroute MHC-I to the trans-Golgi network (TGN) in a phosphofurin acidic cluster sorting protein (PACS) 1, adaptor protein (AP)-1, and clathrin-dependent manner. More recently, an alternative model has been proposed, in which Nef uses AP-1 to direct MHC-I to endosomes and lysosomes. Here, we show that knocking down either AP-1 or clathrin with small interfering RNA inhibits the down-regulation of HLA-A2 (an MHC-I isotype) by Nef in HeLa cells. However, knocking down PACS-1 has no effect, not only on Nef-induced down-regulation of HLA-A2 but also on the localization of other proteins containing acidic cluster motifs. Surprisingly, knocking down AP-2 actually enhances Nef activity. Immuno-electron microscopy labeling of Nef-expressing cells indicates that HLA-A2 is rerouted not to the TGN, but to endosomes. In AP-2-depleted cells, more of the HLA-A2 localizes to the inner vesicles of multivesicular bodies. We propose that depleting AP-2 potentiates Nef activity by altering the membrane composition and dynamics of endosomes and causing increased delivery of HLA-A2 to a prelysosomal compartment.     

Biogenesis of the multifunctional lipid droplet: Lipids,proteins, and sites

The actin filament severing protein cofilin-1 (CFL-1) is required for actin and P-type ATPase secretory pathway calcium ATPase (SPCA)-dependent sorting of secretory proteins at the trans-Golgi network (TGN). How these proteins interact and activate the pump to facilitate cargo sorting, however, is not known. We used purified proteins to assess interaction of the cytoplasmic domains of SPCA1 with actin and CFL-1. A 132–amino acid portion of the SPCA1 phosphorylation domain (P-domain) interacted with actin in a CFL-1–dependent manner. This domain, coupled to nickel nitrilotriacetic acid (Ni-NTA) agarose beads, specifically recruited F-actin in the presence of CFL-1 and, when expressed in HeLa cells, inhibited Ca²⁺ entry into the TGN and secretory cargo sorting. Mutagenesis of four amino acids in SPCA1 that represent the CFL-1 binding site also affected Ca²⁺ import into the TGN and secretory cargo sorting. Altogether, our findings reveal the mechanism of CFL-1–dependent recruitment of actin to SPCA1 and the significance of this interaction for Ca²⁺ influx and secretory cargo sorting.     

Intracellular trafficking of LRP9 is dependent on two acidic cluster/dileucine motifs

LDL receptor-related protein 9 (LRP9) is a distant member of the low-density lipoprotein receptor (LDLR) superfamily. To date, there are no reports on the cellular distribution of LRP9 or the signals responsible for its localization. Here, we investigated the intracellular localization and trafficking of LRP9. Using confocal microscopy, we demonstrated that LRP9 was not present at the plasma membrane but co-localized with various markers of the trans-Golgi network (TGN) and endosomes. This co-localization was dependent on the presence of two acidic cluster/dileucine (DXXLL) motifs in the cytoplasmic tail of LRP9, which interact with GGA proteins, clathrin adaptors involved in transport between the TGN and endosomes. LRP9 is the first example of a transmembrane protein with an internal GGA-binding sequence in addition to the usual C-terminal motif. An inactivating mutation (LL --> AA) in both DXXLL motifs, which completely inhibited the interaction of LRP9 with GGA proteins, led to an intracellular redistribution of LRP9 from the TGN to early endosomes and the cell surface, indicating that the two DXXLL motifs are essential sorting determinants of LRP9. In conclusion, our results suggest that LRP9 cycles between the TGN, endosomes and the plasma membrane through a GGA dependent-trafficking mechanism.     

Regulation of Endosome Sorting by a Specific PP2A Isoform

The regulated sorting of proteins within the trans-Golgi network (TGN)/endosomal system is a key determinant of their biological activity in vivo. For example, the endoprotease furin activates of a wide range of proproteins in multiple compartments within the TGN/endosomal system. Phosphorylation of its cytosolic domain by casein kinase II (CKII) promotes the localization of furin to the TGN and early endosomes whereas dephosphorylation is required for efficient transport between these compartments (Jones, B.G., L. Thomas, S.S. Molloy, C.D. Thulin, M.D. Fry, K.A. Walsh, and G. Thomas. 1995. EMBO [Eur. Mol. Biol. Organ.] J. 14:5869–5883). Here we show that phosphorylated furin molecules internalized from the cell surface are retained in a local cycling loop between early endosomes and the plasma membrane. This cycling loop requires the phosphorylation state-dependent furin-sorting protein PACS-1, and mirrors the trafficking pathway described recently for the TGN localization of furin (Wan, L., S.S. Molloy, L. Thomas, G. Liu, Y. Xiang, S.L. Ryback, and G. Thomas. 1998. Cell. 94:205–216). We also demonstrate a novel role for protein phosphatase 2A (PP2A) in regulating protein localization in the TGN/endosomal system. Using baculovirus recombinants expressing individual PP2A subunits, we show that the dephosphorylation of furin in vitro requires heterotrimeric phosphatase containing B family regulatory subunits. The importance of this PP2A isoform in directing the routing of furin from early endosomes to the TGN was established using SV-40 small t antigen as a diagnostic tool in vivo. The role of both CKII and PP2A in controlling multiple sorting steps in the TGN/endosomal system indicates that the distribution of itinerant membrane proteins may be acutely regulated via signal transduction pathways.     

The acidic cluster of the CK2 site of the cation-dependent mannose 6-phosphate receptor (CD-MPR) but not its phosphorylation is required for GGA1 and AP-1 binding

Lysosomal biogenesis depends on proper transport of lysosomal enzymes by the cation-dependent mannose 6-phosphate receptor (CD-MPR) from the trans-Golgi network (TGN) to endosomes. Trafficking of the CDMPR is mediated by sorting signals in its cytoplasmic tail. GGA1 (Golgi-localizing, gamma-ear-containing, ARF-binding protein-1) binds to CD-MPR in the TGN and targets the receptor to clathrin-coated pits for transport from the TGN to endosomes. The motif of the CD-MPR that interacts with GGA1 was shown to be 61DXXLL65. Reports on increased affinity of cargo, when phosphorylated by casein kinase 2 (CK2), to GGAs focused our interest on the effect of the CD-MPR CK2 site on binding to GGA1. Here we demonstrate that Glu58 and Glu59 of the CK2 site are essential for high affinity GGA1 binding in vitro, whereas the phosphorylation of Ser57 of the CD-MPR has no influence on receptor binding to GGA1. Furthermore, the in vivo interaction between GGA1 and CD-MPR was abolished only when all residues involved in GGA1 binding were mutated, namely, Glu58, Glu59, Asp61, Leu64, and Leu65. In contrast, the binding of adaptor protein-1 (AP-1) to CD-MPR required all the glutamates surrounding the phosphorylation site, namely, Glu55, Glu56, Glu58, and Glu59, but like GGA1 binding, was independent of the phosphorylation of Ser57. The binding affinity of GGA1 to the CD-MPR was found to be 2.4-fold higher than that of AP-1. This could regulate the binding of the two proteins to the partly overlapping sorting signals, allowing AP-1 binding to the CD-MPR only when GGA1 is released upon autoinhibition by phosphorylation.     

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) protein-induced lysosomal translocation of proapoptotic effectors is mediated by phosphofurin acidic cluster sorting protein-2 (PACS-2)

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-induced apoptosis of liver cancer cell lines requires death receptor-5 (DR5)-dependent permeabilization of lysosomal membranes. Ligated DR5 triggers recruitment of the proapoptotic proteins Bim and Bax to lysosomes, releasing cathepsin B into the cytosol where it mediates mitochondria membrane permeabilization and activation of executioner caspases. Despite the requirement for lysosome membrane permeabilization during TRAIL-induced apoptosis, little is known about the mechanism that controls recruitment of Bim and Bax to lysosomal membranes. Here we report that TRAIL induces recruitment of the multifunctional sorting protein phosphofurin acidic cluster sorting protein-2 (PACS-2) to DR5-positive endosomes in Huh-7 cells where it forms an immunoprecipitatable complex with Bim and Bax on lysosomal membranes. shRNA-targeted knockdown of PACS-2 prevents recruitment of Bim or Bax to lysosomes, blunting the TRAIL-induced lysosome membrane permeabilization. Consistent with the reduced lysosome membrane permeabilization, shRNA knockdown of PACS-2 in Huh-7 cells reduced TRAIL-induced apoptosis and increased clonogenic cell survival. The determination that recombinant PACS-2 bound Bim but not Bax in vitro and that shRNA knockdown of Bim blocked Bax recruitment to lysosomes suggests that TRAIL/DR5 triggers endosomal PACS-2 to recruit Bim and Bax to lysosomes to release cathepsin B and induce apoptosis. Together, these findings provide insight into the lysosomal pathway of apoptosis.     

Sorting of plant vacuolar proteins is initiated in the ER

Transport of soluble cargo molecules to the lytic vacuole of plants requires vacuolar sorting receptors (VSRs) to divert transport of vacuolar cargo from the default secretory route to the cell surface. Just as important is the trafficking of the VSRs themselves, a process that encompasses anterograde transport of receptor–ligand complexes from a donor compartment, dissociation of these complexes upon arrival at the target compartment, and recycling of the receptor back to the donor compartment for a further round of ligand transport. We have previously shown that retromer‐mediated recycling of the plant VSR BP80 starts at the trans‐Golgi network (TGN). Here we demonstrate that inhibition of retromer function by either RNAi knockdown of sorting nexins (SNXs) or co‐expression of mutants of SNX1/2a specifically inhibits the ER export of VSRs as well as soluble vacuolar cargo molecules, but does not influence cargo molecules destined for the COPII‐mediated transport route. Retention of soluble cargo despite ongoing COPII‐mediated bulk flow can only be explained by an interaction with membrane‐bound proteins. Therefore, we examined whether VSRs are capable of binding their ligands in the lumen of the ER by expressing ER‐anchored VSR derivatives. These experiments resulted in drastic accumulation of soluble vacuolar cargo molecules in the ER. This demonstrates that the ER, rather than the TGN, is the location of the initial VSR–ligand interaction. It also implies that the retromer‐mediated recycling route for the VSRs leads from the TGN back to the ER.     

The tyrosine-based lysosomal targeting signal in lamp-1 mediates sorting into Golgi-derived clathrin-coated vesicles.

Diversion of membrane proteins from the trans-Golgi network (TGN) or the plasma membrane into the endosomal system occurs via clathrin-coated vesicles (CCVs). These sorting events may require the interaction of cytosolic domain signals with clathrin adaptor proteins (APs) at the TGN (AP-1) or the plasma membrane (AP-2). While tyrosine- and di-leucine-based signals in several proteins mediate endocytosis via cell surface CCVs, segregation into Golgi-derived CCVs has so far only been documented for the mannose 6-phosphate receptors, where it is thought to require a casein kinase II phosphorylation site adjacent to a di-leucine motif. Although recently tyrosine-based signals have also been shown to interact with the mu chain of AP-1 in vitro, it is not clear if these signals also bind intact AP-1 adaptors, nor if they can mediate sorting of proteins into AP-1 CCVs. Here we show that the cytosolic domain of the lysosomal membrane glycoprotein lamp-1 binds AP-1 and AP-2. Furthermore, lamp-1 is present in AP-1-positive vesicles and tubules in the trans-region on the Golgi complex. AP-1 binding as well as localization to AP-1 CCVs require the presence of the functional tyrosine-based lysosomal targeting signal of lamp-1. These results indicate that lamp-1 can exit the TGN in CCVs and that tyrosine signals can mediate these sorting events.