Cytoplasmic determinants involved in direct lysosomal sorting, endocytosis, and basolateral targeting of rat lgp120 (lamp-I) in MDCK cells

Rat lysosomal glycoprotein 120 (lgp120; lamp-I) is a transmembrane protein that is directly delivered from the trans-Golgi network (TGN) to the endosomal/lysosomal system without prior appearance on the cell surface. Its short cytosolic domain of 11 residues encodes determinants for direct lysosomal sorting, endocytosis and, in polarized cells, basolateral targeting. We now characterize the structural requirements in the cytosolic domain required for sorting of lgp120 into the different pathways. Our results show that the cytoplasmic tail is sufficient to mediate direct transport from the trans-Golgi network (TGN) to lysosomes and that a G7-Y8-X-X-I11 motif is crucial for this sorting event. While G7 is only critical for direct lysosomal sorting in the TGN, Y8 and I11 are equally important for lysosomal sorting, endocytosis, and basolateral targeting. Thus, a small motif of five amino acids in the cytoplasmic tail of lgp120 can be recognized by the sorting machinery at several cellular locations and direct the protein into a variety of intracellular pathways.     

Identification and Targeting of an Interaction between a Tyrosine Motif within Hepatitis C Virus Core Protein and AP2M1 Essential for Viral Assembly

Novel therapies are urgently needed against hepatitis C virus infection (HCV), a major global health problem. The current model of infectious virus production suggests that HCV virions are assembled on or near the surface of lipid droplets, acquire their envelope at the ER, and egress through the secretory pathway. The mechanisms of HCV assembly and particularly the role of viral-host protein-protein interactions in mediating this process are, however, poorly understood. We identified a conserved heretofore unrecognized YXXΦ motif (Φ is a bulky hydrophobic residue) within the core protein. This motif is homologous to sorting signals within host cargo proteins known to mediate binding of AP2M1, the μ subunit of clathrin adaptor protein complex 2 (AP-2), and intracellular trafficking. Using microfluidics affinity analysis, protein-fragment complementation assays, and co-immunoprecipitations in infected cells, we show that this motif mediates core binding to AP2M1. YXXΦ mutations, silencing AP2M1 expression or overexpressing a dominant negative AP2M1 mutant had no effect on HCV RNA replication, however, they dramatically inhibited intra- and extracellular infectivity, consistent with a defect in viral assembly. Quantitative confocal immunofluorescence analysis revealed that core''s YXXΦ motif mediates recruitment of AP2M1 to lipid droplets and that the observed defect in HCV assembly following disruption of core-AP2M1 binding correlates with accumulation of core on lipid droplets, reduced core colocalization with E2 and reduced core localization to trans-Golgi network (TGN), the presumed site of viral particles maturation. Furthermore, AAK1 and GAK, serine/threonine kinases known to stimulate binding of AP2M1 to host cargo proteins, regulate core-AP2M1 binding and are essential for HCV assembly. Last, approved anti-cancer drugs that inhibit AAK1 or GAK not only disrupt core-AP2M1 binding, but also significantly inhibit HCV assembly and infectious virus production. These results validate viral-host interactions essential for HCV assembly and yield compounds for pharmaceutical development.     

EpsinR: an ENTH domain-containing protein that interacts with AP-1

We have used GST pulldowns from A431 cell cytosol to identify three new binding partners for the gamma-adaptin appendage: Snx9, ARF GAP1, and a novel ENTH domain-containing protein, epsinR. EpsinR is a highly conserved protein that colocalizes with AP-1 and is enriched in purified clathrin-coated vesicles. However, it does not require AP-1 to get onto membranes and remains membrane-associated in AP-1-deficient cells. Moreover, although epsinR binds AP-1 via its COOH-terminal domain, its NH(2)-terminal ENTH domain can be independently recruited onto membranes, both in vivo and in vitro. Brefeldin A causes epsinR to redistribute into the cytosol, and recruitment of the ENTH domain requires GTPgammaS, indicating that membrane association is ARF dependent. In protein-lipid overlay assays, the epsinR ENTH domain binds to PtdIns(4)P, suggesting a possible mechanism for ARF-dependent recruitment onto TGN membranes. When epsinR is depleted from cells by RNAi, cathepsin D is still correctly processed intracellularly to the mature form. This indicates that although epsinR is likely to be an important component of the AP-1 network, it is not necessary for the sorting of lysosomal enzymes.     

The trans-Golgi network accessory protein p56 promotes long-range movement of GGA/clathrin-containing transport carriers and lysosomal enzyme sorting

The sorting of acid hydrolase precursors at the trans-Golgi network (TGN) is mediated by binding to mannose 6-phosphate receptors (MPRs) and subsequent capture of the hydrolase-MPR complexes into clathrin-coated vesicles or transport carriers (TCs) destined for delivery to endosomes. This capture depends on the function of three monomeric clathrin adaptors named GGAs. The GGAs comprise a C-terminal "ear" domain that binds a specific set of accessory proteins. Herein we show that one of these accessory proteins, p56, colocalizes and physically interacts with the three GGAs at the TGN. Moreover, overexpression of the GGAs enhances the association of p56 with the TGN, and RNA interference (RNAi)-mediated depletion of the GGAs decreases the TGN association and total levels of p56. RNAi-mediated depletion of p56 or the GGAs causes various degrees of missorting of the precursor of the acid hydrolase, cathepsin D. In the case of p56 depletion, this missorting correlates with decreased mobility of GGA-containing TCs. Transfection with an RNAi-resistant p56 construct, but not with a p56 construct lacking the GGA-ear-interacting motif, restores the mobility of the TCs. We conclude that p56 tightly cooperates with the GGAs in the sorting of cathepsin D to lysosomes, probably by enabling the movement of GGA-containing TCs.     

The amino acids upstream of NH(2)-terminal dileucine motif play a role in regulating the intracellular sorting of the Class III transporters GLUT8 and GLUT12

Abstract

The transport of glucose across cell membranes is mediated by a family of facilitative glucose transporters (GLUTs). The class III glucose transporters GLUT8 and GLUT12 both contain a similar [DE]XXXL[LI] dileucine sorting signal in their amino terminus. This type of dileucine motif facilitates protein trafficking to various organelles or to the plasma membrane via interactions with adaptor protein (AP) complexes. The [DE]XXXL[LI] motif in GLUT8 is thought to direct it to late endosomal/lysosomal compartments via its interactions with AP1 and AP2. Unlike GLUT8, the [DE]XXXL[LI] motif does not direct GLUT12 to a lysosomal compartment. Rather, GLUT12 resides in the Golgi network and at the plasma membrane. In a previous study, we found that exchanging the XXX (TQP) residues in GLUT8 with the corresponding residues in GLUT12 (GPN) resulted in a dramatic missorting of GLUT8 to the cell surface. We postulated that the XXX amino acids upstream of the dileucine motif in GLUT8 influence the degree of interaction between the [DE]XXXL[LI] motif and adaptor proteins. To further explore its trafficking mechanisms, we created mutant constructs to identify the role that each of the individual XXX amino acids has for regulating the intracellular sorting of GLUT8. Here we find that the XXX amino acids, specifically the position of a proline -2 from the dileucine residues, influence the affinity of APs for GLUT8 and GLUT12.     

The amino acids upstream of NH(2)-terminal dileucine motif play a role in regulating the intracellular sorting of the Class III transporters GLUT8 and GLUT12

The transport of glucose across cell membranes is mediated by a family of facilitative glucose transporters (GLUTs). The class III glucose transporters GLUT8 and GLUT12 both contain a similar [DE]XXXL[LI] dileucine sorting signal in their amino terminus. This type of dileucine motif facilitates protein trafficking to various organelles or to the plasma membrane via interactions with adaptor protein (AP) complexes. The [DE]XXXL[LI] motif in GLUT8 is thought to direct it to late endosomal/lysosomal compartments via its interactions with AP1 and AP2. Unlike GLUT8, the [DE]XXXL[LI] motif does not direct GLUT12 to a lysosomal compartment. Rather, GLUT12 resides in the Golgi network and at the plasma membrane. In a previous study, we found that exchanging the XXX (TQP) residues in GLUT8 with the corresponding residues in GLUT12 (GPN) resulted in a dramatic missorting of GLUT8 to the cell surface. We postulated that the XXX amino acids upstream of the dileucine motif in GLUT8 influence the degree of interaction between the [DE]XXXL[LI] motif and adaptor proteins. To further explore its trafficking mechanisms, we created mutant constructs to identify the role that each of the individual XXX amino acids has for regulating the intracellular sorting of GLUT8. Here we find that the XXX amino acids, specifically the position of a proline -2 from the dileucine residues, influence the affinity of APs for GLUT8 and GLUT12.     

Requirement of the human GARP complex for mannose 6-phosphate-receptor-dependent sorting of cathepsin D to lysosomes

The biosynthetic sorting of acid hydrolases to lysosomes relies on transmembrane, mannose 6-phosphate receptors (MPRs) that cycle between the TGN and endosomes. Herein we report that maintenance of this cycling requires the function of the mammalian Golgi-associated retrograde protein (GARP) complex. Depletion of any of the three GARP subunits, Vps52, Vps53, or Vps54, by RNAi impairs sorting of the precursor of the acid hydrolase, cathepsin D, to lysosomes and leads to its secretion into the culture medium. As a consequence, lysosomes become swollen, likely due to a buildup of undegraded materials. Missorting of cathepsin D in GARP-depleted cells results from accumulation of recycling MPRs in a population of light, small vesicles downstream of endosomes. These vesicles might correspond to intermediates in retrograde transport from endosomes to the TGN. Depletion of GARP subunits also blocks the retrograde transport of the TGN protein, TGN46, and the B subunit of Shiga toxin. These observations indicate that the mammalian GARP complex plays a general role in the delivery of retrograde cargo into the TGN. We also report that a Vps54 mutant protein in the Wobbler mouse strain is active in retrograde transport, thus explaining the viability of these mutant mice.     

Recognition of dileucine-based sorting signals from HIV-1 Nef and LIMP-II by the AP-1 gamma-sigma1 and AP-3 delta-sigma3 hemicomplexes

The sorting of transmembrane proteins to endosomes and lysosomes is mediated by signals present in the cytosolic tails of the proteins. A subset of these signals conform to the [DE]XXXL[LI] consensus motif and mediate sorting via interactions with heterotetrameric adaptor protein (AP) complexes. However, the identity of the AP subunits that recognize these signals remains controversial. We have used a yeast three-hybrid assay to demonstrate that [DE]XXXL[LI]-type signals from the human immunodeficiency virus negative factor protein and the lysosomal integral membrane protein II interact with combinations of the gamma and sigma1 subunits of AP-1 and the delta and sigma3 subunits of AP-3, but not the analogous combinations of AP-2 and AP-4 subunits. The sequence requirements for these interactions are similar to those for binding to the whole AP complexes in vitro and for function of the signals in vivo. These observations reveal a novel mode of recognition of sorting signals involving the gamma/delta and sigma subunits of AP-1 and AP-3.     

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.     

The ubiquitin ligase RNF126 regulates the retrograde sorting of the cation-independent mannose 6-phosphate receptor

The ubiquitin proteasome system is central to the regulation of a number of intracellular sorting pathways in mammalian cells including quality control at the endoplasmic reticulum and the internalization and endosomal sorting of cell surface receptors. Here we describe that RNF126, an E3 ubiquitin ligase, is involved in the sorting of the cation-independent mannose 6-phosphate receptor (CI-MPR). In cells transiently depleted of RNF126, the CI-MPR is dispersed into Rab4 positive endosomes and the efficiency of retrograde sorting is delayed. Furthermore, the stable knockdown of RNF126 leads to the lysosomal degradation of CI-MPR and missorting of cathepsin D. RNF126 specifically regulates the sorting of the CI-MPR as other cargo that follow the retrograde sorting route including the cholera toxin, furin and TGN38 are unaffected in the absence of RNF126. Lastly we show that the RING finger domain of RNF126 is required to rescue the decrease in CI-MPR levels, suggesting that the ubiquitin ligase activity of RNF126 is required for CI-MPR sorting. Together, our data indicate that the ubiquitin ligase RNF126 has a role in the retrograde sorting of the CI-MPR     

The dileucine motif within the tail of MPR46 is required for sorting of the receptor in endosomes

The cytoplasmic tail of MPR46 carries a leucine-based motif that is required for the sorting of lysosomal enzymes by the receptor. In addition, it is one of three independent, but functionally redundant, internalization signals present in the cytoplasmic tail of MPR46. We have analyzed a mutant of MPR46, in which the dileucine pair was replaced by alanines (MPR46 LL/AA) with respect to its intracellular distribution and trafficking. Ultrastructural analysis of cells expressing the MPR46 LL/AA mutant revealed that the substitution of the dileucine pair causes a shift of the receptor distribution from the TGN, where it is packaged into AP1-containing vesicles, to vesicular structures distributed throughout the cytoplasm. The vesicles could be identified as early endosomes with internalized BSA-gold and rab5 as markers. By analyzing the receptor trafficking biochemically, we found that return of the LL/AA mutant receptor from the plasma membrane/endosome pool back to the TGN was impaired, while recycling from endosomes to the plasma membrane was enhanced. In conclusion, our data indicate that the dileucine motif in the MPR46 tail is required for a sorting event in endosomes.     

Sorting of encystation-specific cysteine protease to lysosome-like peripheral vacuoles in Giardia lamblia requires a conserved tyrosine-based motif

Encystation-specific cysteine protease (ESCP) was the first membrane-associated protein described to be part of the lysosome-like peripheral vacuoles in the intestinal parasite Giardia lamblia. ESCP is homologous to cathepsin C enzymes of higher eukaryotes, but is distinguished from other lysosomal cysteine proteases because it possesses a transmembrane domain and a short cytoplasmic tail. Tyrosine-based motifs within tails of membrane proteins are known to participate in endosomal/lysosomal protein sorting in higher eukaryotes. In this study, we show that a YRPI motif within the ESCP cytoplasmic tail is necessary and sufficient to mediate ESCP sorting to peripheral vacuoles in Giardia. Deletion and point mutation analysis demonstrated that the tyrosine residue is critical for ESCP sorting, whereas amino acids located at the Y+1 (Arg), Y+2 (Pro), and Y+3 (Ile) positions show minimal effect. Loss of the motif resulted in surface localization, whereas addition of the motif to a variant-specific surface protein resulted in lysosomal localization. Although Giardia trophozoites lack a morphologically discernible Golgi apparatus, our findings indicate that this parasite directs proteins to the lysosomes using a conserved sorting signal similar to that used by yeast and mammalian cells. Because Giardia is one of the earliest branching protist, these results demonstrate that sorting motifs for specific protein traffic developed very early during eukaryotic evolution.     

GGA proteins bind ubiquitin to facilitate sorting at the trans-Golgi network

Ubiquitination functions as a sorting signal for lysosomal degradation of cell-surface proteins by facilitating their internalization from the plasma membrane and incorporation into lumenal vesicles of multivesicular bodies (MVBs). Ubiquitin may also mediate sorting of proteins from the trans-Golgi network (TGN) to the endosome, thereby preventing their appearance on the cell surface and hastening their degradation in the lysosome-vacuole. Substantiation of a direct ubiquitin-dependent TGN sorting pathway relies in part on identifying candidate machinery that may function as a ubiquitin-sorting 'receptor'at the TGN. Members of the GGA family of coat proteins localize to the TGN and promote the incorporation of proteins into clathrin-coated vesicles destined for transport to endosomes. We show that the GGA coat proteins bind directly to ubiquitin through their GAT domain and demonstrate that this interaction is required for the ubiquitin-dependent sorting of the Gap1 amino acid transporter from the TGN to endosomes. Thus, GGA proteins fulfill the role of ubiquitin sorting receptors at the TGN.     

Role of an acidic cluster/dileucine motif in cation-independent mannose 6-phosphate receptor traffic

The endocytic trafficking of the cation-independent mannose 6-phosphate receptor (CI-MPR) involves multiple sorting steps. A cluster of acidic amino acids followed by a dileucine motif in the cytoplasmic tail has been proposed to mediate receptor sorting from the trans Golgi network (TGN) to late endosomes. Mutations in this motif impair lysosomal enzyme sorting by preventing association of CI-MPR with coat proteins. The role of the acidic cluster/dileucine motif in the post-endocytic transport of the receptor was examined using the CI-MPR mutants, AC01 and D160E (Chen HJ, Yuan J, Lobel P. J Biol Chem 1997;272:7003-7012). Following internalization, wild type (WT) CI-MPR is transported through sorting endosomes into the endocytic recycling compartment (ERC), after which it traffics to the TGN and other organelles. However, the mutants localize mostly to the ERC and only a small portion reaches the TGN, suggesting that the sorting of the CI-MPR mutants from the ERC into the TGN is severely impaired. We observed no defect in receptor internalization or in the rate of tail mutant recycling to the cell surface compared to the WT. These results demonstrate that the acidic cluster/dileucine motif of CI-MPR is critical for receptor sorting at early stages of intracellular transport following endocytosis.     

PI4P promotes the recruitment of the GGA adaptor proteins to the trans-Golgi network and regulates their recognition of the ubiquitin sorting signal

Phosphatidylinositol 4 phosphate (PI4P) is highly enriched in the trans-Golgi network (TGN). Here we establish that PI4P is a key regulator of the recruitment of the GGA clathrin adaptor proteins to the TGN and that PI4P has a novel role in promoting their recognition of the ubiquitin (Ub) sorting signal. Knockdown of PI4KIIalpha by RNA interference (RNAi), which depletes the TGN's PI4P, impaired the recruitment of the GGAs to the TGN. GGAs bind PI4P primarily through their GAT domain, in a region called C-GAT, which also binds Ub but not Arf1. We identified two basic residues in the GAT domain that are essential for PI4P binding in vitro and for the recruitment of GGAs to the TGN in vivo. Unlike wild-type GGA, GGA with mutated GATs failed to rescue the abnormal TGN phenotype of the GGA RNAi-depleted cells. These residues partially overlap with those that bind Ub, and PI4P increased the affinity of the GAT domain for Ub. Because the recruitment of clathrin adaptors and their cargoes to the TGN is mediated through a web of low-affinity interactions, our results show that the dual roles of PI4P can promote specific GGA targeting and cargo recognition at the TGN.     

The biogenesis of the MHC class II compartment in human I-cell disease B lymphoblasts

The localization and intracellular transport of major histocompatibility complex (MHC) class II molecules nd lysosomal hydrolases were studied in I-Cell Disease (ICD) B lymphoblasts, which possess a mannose 6-phosphate (Man-6-P)-independent targeting pathway for lysosomal enzymes. In the trans-Golgi network (TGN), MHC class II- invariant chain complexes colocalized with the lysosomal hydrolase cathepsin D in buds and vesicles that lacked markers of clathrin-coated vesicle-mediated transport. These vesicles fused with the endocytic pathway leading to the formation of "early" MHC class II-rich compartments (MIICs). Similar structures were observed in the TGN of normal beta lymphoblasts although they were less abundant. Metabolic labeling and subcellular fractionation experiments indicated that newly synthesized cathepsin D and MHC class II-invariant chain complexes enter a non-clathrin-coated vesicular structure after their passage through the TGN and segregation from the secretory pathway. These vesicles were also devoid of the cation-dependent mannose 6-phosphate (Man-6-P) receptor, a marker of early and late endosomes. These findings suggest that in ICD B lymphoblasts the majority of MHC class II molecules are transported directly from the TGN to "early" MIICs and that acid hydrolases cam be incorporated into MIICs simultaneously by a Man-6-P-independant process.     

Role for phosphatidylinositol 3-kinase in the sorting and transport of newly synthesized lysosomal enzymes in mammalian cells

Previous work with the yeast Saccharomyces cerevisiae has demonstrated a role for a phosphatidylinositol-specific PI 3-kinase, the product of the VPS34 gene, in the targeting of newly synthesized proteins to the vacuole, an organelle functionally equivalent to mammalian lysosomes (Schu, P. V., K. Takegawa, M. J. Fry, J. H. Stack, M. D. Waterfield, and S. D. Emr. 1993. Science [Wash. DC]. 260:88-91). The activity of Vps34p kinase is significantly reduced by the PI 3-kinase inhibitors wortmannin, a fungal metabolite, and LY294002, a quercetin analog (Stack, J. H., and S. D. Emr. 1994. J. Biol. Chem. 269:31552-31562). We show here that at concentrations which inhibit VPS34-encoded PI 3- kinase activity, wortmannin also inhibits the processing and delivery of newly synthesized cathepsin D to lysosomes in mammalian cells with half-maximal inhibition of delivery occurring at 100 nM wortmannin. As a result of wortmannin action, newly synthesized, unprocessed cathepsin D is secreted into the media. Moreover, after accumulation in the trans- Golgi network (TGN) at 20 degrees C, cathepsin D was rapidly missorted to the secretory pathway after addition of wortmannin and shifting to 37 degrees C. At concentrations that inhibited lysosomal enzyme delivery, both wortmannin and LY294002 caused a highly specific dilation of mannose 6-phosphate receptor (M6PR)-enriched vesicles of the prelysosome compartment (PLC), which swelled to approximately 1 micron within 15 min after treatment. With increasing time, the inhibitors caused a significant yet reversible change in M6PR distribution. By 3 h of treatment, the swollen PLC vacuoles were essentially depleted of receptors and, in addition, there was a fourfold loss of receptors from the cell surface. However, M6PRs were still abundant in the TGN. These results are most consistent with the interpretation that PI 3-kinase regulates the trafficking of lysosomal enzymes by interfering with a M6PR-dependent sorting event in the TGN. Moreover, they provide evidence that trafficking of soluble hydrolases to mammalian lysosomes and yeast vacuoles rely on similar regulatory mechanisms.     

Direct interaction of the trans-Golgi network membrane protein, TGN38, with the F-actin binding protein, neurabin.

TGN38 is a type I integral membrane protein that constitutively cycles between the trans-Golgi network (TGN) and plasma membrane. The cytosolic domain of TGN38 interacts with AP2 clathrin adaptor complexes via the tyrosine-containing motif (-SDYQRL-) to direct internalization from the plasma membrane. This motif has previously been shown to direct both internalization and subsequent TGN targeting of TGN38. We have used the cytosolic domain of TGN38 in a two-hybrid screen, and we have identified the brain-specific F-actin binding protein neurabin-I as a TGN38-binding protein. We demonstrate a direct interaction between TGN38 and the ubiquitous homologue of neurabin-I, neurabin-II (also called spinophilin). We have used a combination of yeast two-hybrid and in vitro protein interaction assays to show that this interaction is dependent on the serine (but not tyrosine) residue of the known TGN38 trafficking motif. We show that TGN38 interacts with the coiled coil region of neurabin in vitro and binds preferentially with the dimeric form of neurabin. TGN38 and neurabin also interact in vivo as demonstrated by coimmunoprecipitation from stably transfected PC12 cells. These data suggest that neurabin provides a direct physical link between TGN38-containing membranes and the actin cytoskeleton.     

Vacuolar Protein Sorting Receptor in Giardia lamblia

In Giardia, lysosome-like peripheral vacuoles (PVs) need to specifically coordinate their endosomal and lysosomal functions to be able to successfully perform endocytosis, protein degradation and protein delivery, but how cargo, ligands and molecular components generate specific routes to the PVs remains poorly understood. Recently, we found that delivering membrane Cathepsin C and the soluble acid phosphatase (AcPh) to the PVs is adaptin (AP1)-dependent. However, the receptor that links AcPh and AP1 was never described. We have studied protein-binding to AcPh by using H6-tagged AcPh, and found that a membrane protein interacted with AcPh. This protein, named GlVps (for Giardia lamblia Vacuolar protein sorting), mainly localized to the ER-nuclear envelope and in some PVs, probably functioning as the sorting receptor for AcPh. The tyrosine-binding motif found in the C-terminal cytoplasmic tail domain of GlVps was essential for its exit from the endoplasmic reticulum and transport to the vacuoles, with this motif being necessary for the interaction with the medium subunit of AP1. Thus, the mechanism by which soluble proteins, such as AcPh, reach the peripheral vacuoles in Giardia appears to be very similar to the mechanism of lysosomal protein-sorting in more evolved eukaryotic cells.     

Polarized Traffic of LRP1 Involves AP1B and SNX17 Operating on Y-dependent Sorting Motifs in Different Pathways

Low-density lipoprotein receptor–related protein 1 (LRP1) is an endocytic recycling receptor with two cytoplasmic tyrosine-based basolateral sorting signals. Here we show that during biosynthetic trafficking LRP1 uses AP1B adaptor complex to move from a post-TGN recycling endosome (RE) to the basolateral membrane. Then it recycles basolaterally from the basolateral sorting endosome (BSE) involving recognition by sorting nexin 17 (SNX17). In the biosynthetic pathway, Y₂₉ but not N₂₆ from a proximal NPXY directs LRP1 basolateral sorting from the TGN. A N₂₆A mutant revealed that this NPXY motif recognized by SNX17 is required for the receptor's exit from BSE. An endocytic Y₆₃ATL₆₆ motif also functions in basolateral recycling, in concert with an additional endocytic motif (LL_86,87), by preventing LRP1 entry into the transcytotic apical pathway. All this sorting information operates similarly in hippocampal neurons to mediate LRP1 somatodendritic distribution regardless of the absence of AP1B in neurons. LRP1 basolateral distribution results then from spatially and temporally segregation steps mediated by recognition of distinct tyrosine-based motifs. We also demonstrate a novel function of SNX17 in basolateral/somatodendritic recycling from a different compartment than AP1B endosomes.