Zinc is required for structural stability of the C-terminus of archaeal translation initiation factor aIF2beta

We report that the Vps10p domain receptor sorLA binds the adaptor proteins GGA1 and -2, which take part in Golgi-endosome sorting. The GGAs bind with differential requirements via three critical residues in the C-terminal segment of the sorLA cytoplasmic tail. Unlike in sortilin and the mannose 6-phosphate receptors, the GGA-binding segment in sorLA contains neither an acidic cluster nor a dileucine. Our results support the concept of sorLA as a potential sorting receptor and suggest that key residues in sorLA and sortilin conform to a new type of motif (psi-psi-X-X-phi) defining minimum requirements for GGA binding to cytoplasmic receptor domains.     

The mannose 6-phosphate receptor cytoplasmic domain is not sufficient to alter the cellular distribution of a chimeric EGF receptor.

Unlike most receptors, 300 kd mannose 6-phosphate receptors (MPRs) are localized primarily in the trans-Golgi network (TGN) and endosomes, and they cycle constitutively between these compartments. Yet, when present at the cell surface, MPRs are internalized together with other cell surface receptors in clathrin-coated vesicles. We constructed a chimeric receptor, comprised of human EGF receptor extracellular and transmembrane domains joined to the bovine MPR cytoplasmic domain, to test whether the MPR cytoplasmic domain contained sufficient information to direct a cell surface receptor into both of these transport pathways. The expressed protein was stable, bound EGF with high affinity, and was efficiently endocytosed and recycled back to the cell surface, in the presence or absence of EGF. If the cytoplasmic domain alone is responsible for sorting native MPRs, chimeric receptors might have been expected to be located primarily in the TGN and in endosomes at steady state. Surprisingly, under conditions in which essentially all endogenous MPRs were intracellular, greater than 85% of the chimeric receptors were located at the cell surface. These experiments demonstrate that the MPR cytoplasmic domain is not sufficient to alter the distribution of the EGF receptor, and suggest a role for extracellular and transmembrane domains in MPR routing.     

TGN38 recycles basolaterally in polarized Madin-Darby canine kidney cells.

Sorting of newly synthesized plasma membrane proteins to the apical or basolateral surface domains of polarized cells is currently thought to take place within the trans-Golgi network (TGN). To explore the relationship between protein localization to the TGN and sorting to the plasma membrane in polarized epithelial cells, we have expressed constructs encoding the TGN marker, TGN38, in Madin-Darby canine kidney (MDCK) cells. We report that TGN38 is predominantly localized to the TGN of these cells and recycles via the basolateral membrane. Analyses of the distribution of Tac-TGN38 chimeric proteins in MDCK cells suggest that the cytoplasmic domain of TGN38 has information leading to both TGN localization and cycling through the basolateral surface. Mutations of the cytoplasmic domain that disrupt TGN localization also lead to nonpolarized delivery of the chimeric proteins to both surface domains. These results demonstrate an apparent equivalence of basolateral and TGN localization determinants and support an evolutionary relationship between TGN and plasma membrane sorting processes.     

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.     

The phosphorylation state of an autoregulatory domain controls PACS-1-directed protein traffic

PACS-1 is a cytosolic sorting protein that directs the localization of membrane proteins in the trans-Golgi network (TGN)/endosomal system. PACS-1 connects the clathrin adaptor AP-1 to acidic cluster sorting motifs contained in the cytoplasmic domain of cargo proteins such as furin, the cation-independent mannose-6-phosphate receptor and in viral proteins such as human immunodeficiency virus type 1 Nef. Here we show that an acidic cluster on PACS-1, which is highly similar to acidic cluster sorting motifs on cargo molecules, acts as an autoregulatory domain that controls PACS-1-directed sorting. Biochemical studies show that Ser278 adjacent to the acidic cluster is phosphorylated by CK2 and dephosphorylated by PP2A. Phosphorylation of Ser278 by CK2 or a Ser278-->Asp mutation increased the interaction between PACS-1 and cargo, whereas a Ser278-->Ala substitution decreased this interaction. Moreover, the Ser278-->Ala mutation yields a dominant-negative PACS-1 molecule that selectively blocks retrieval of PACS-1-regulated cargo molecules to the TGN. These results suggest that coordinated signaling events regulate transport within the TGN/endosomal system through the phosphorylation state of both cargo and the sorting machinery.     

The sortilin cytoplasmic tail conveys Golgi-endosome transport and binds the VHS domain of the GGA2 sorting protein

Sortilin belongs to a growing family of multiligand type-1 receptors with homology to the yeast receptor Vps10p. Based on structural features and sortilin's intracellular predominance, we have proposed it to be a sorting receptor for ligands in the synthetic pathway as well as on the cell membrane. To test this hypothesis we examine here the cellular trafficking of chimeric receptors containing constructs of the sortilin tail. We report that sorting signals conforming to YXX and dileucine motifs mediate rapid endocytosis of sortilin chimeras, which subsequently travel to the trans-Golgi network, showing little or no recycling. Furthermore, we found that cation-independent mannose 6-phosphate receptor (MPR300)-sortilin chimeras, expressed in mannose 6-phosphate receptor knockout cells, were almost as efficient as MPR300 itself for transport of newly synthesized beta-hexosaminidase and beta-glucuronidase to lysosomes, and established that the sortilin tail contains potent signals for Golgi-endosome sorting. Finally, we provide evidence suggesting that sortilin is the first example of a mammalian receptor targeted by the recently described GGA family of cytosolic sorting proteins, which condition the Vps10p-mediated sorting of yeast carboxypeptidase Y.     

The yeast Vps10p cytoplasmic tail mediates lysosomal sorting in mammalian cells and interacts with human GGAs.

Yeast Vps10p is a receptor for transport of the soluble vacuolar hydrolase carboxypeptidase Y to the lysosome-like vacuole. Its functional equivalents in mammalian cells are the mannose 6-phosphate receptors that mediate sorting to lysosomes of mannose 6-phosphate-containing lysosomal proteins. A chimeric receptor was constructed by substituting the cytoplasmic domain of M(r) 300,000 mannose 6-phosphate receptor with the Vps10p cytoplasmic tail. Expression of the chimera in cells lacking endogenous mannose 6-phosphate receptors resulted in a subcellular receptor distribution and an efficiency in sorting of lysosomal enzymes similar to that of the wild type M(r) 300,000 mannose 6-phosphate receptor. Moreover, the cytoplasmic tail of the Vps10p was found to interact with GGA1 and GGA2, two mammalian members of a recently discovered family of clathrin-binding cytosolic proteins that participate in trans-Golgi network-endosome trafficking in both mammals and yeast. Our findings suggest a conserved machinery for Golgi-endosome/vacuole sorting and may serve as a model for future studies of yeast proteins.     

Interactions of GGA3 with the ubiquitin sorting machinery

The Golgi-localized, gamma-ear-containing, Arf-binding (GGA) proteins constitute a family of clathrin adaptors that are mainly associated with the trans-Golgi network (TGN) and mediate the sorting of mannose 6-phosphate receptors. This sorting is dependent on the interaction of the VHS domain of the GGAs with acidic-cluster-dileucine signals in the cytosolic tails of the receptors. Here we demonstrate the existence of another population of GGAs that are associated with early endosomes. RNA interference (RNAi) of GGA3 expression results in accumulation of the cation-independent mannose 6-phosphate receptor and internalized epidermal growth factor (EGF) within enlarged early endosomes. This perturbation impairs the degradation of internalized EGF, a process that is normally dependent on the sorting of ubiquitinated EGF receptors (EGFRs) to late endosomes. Protein interaction analyses show that the GGAs bind ubiquitin. The VHS and GAT domains of GGA3 are responsible for this binding, as well as for interactions with TSG101, a component of the ubiquitin-dependent sorting machinery. Thus, GGAs may have additional roles in sorting of ubiquitinated cargo.     

ENTH/ANTH domains expand to the Golgi

Clathrin-coated vesicles (CCVs) play important roles in nutrient uptake, downregulation of signaling receptors, pathogen invasion and biogenesis of endosomes and lysosomes. Although detailed models for endocytic CCV formation have emerged, the process of CCV formation at the Golgi and endosomes has been less clear. Key to endocytic CCV formation are proteins containing related phosphoinositide-binding ENTH and ANTH domains. Now, recent studies have identified novel ENTH/ANTH proteins that participate in CCV-mediated traffic between the trans-Golgi Network (TGN) and endosomes and have defined a molecular basis for interaction with AP-1 and GGA adaptors in clathrin coats of the TGN/endosomes. Thus, ENTH/ANTH domain proteins appear to be universal elements in nucleation of clathrin coats.     

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.     

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.     

Localization of TGN38 to the trans-Golgi network: involvement of a cytoplasmic tyrosine-containing sequence

Protein localization to the TGN was investigated by examining the subcellular distribution of chimeric proteins in which the cytoplasmic and/or transmembrane domains of the TGN protein, TGN38, were substituted for the analogous domains of the plasma membrane protein, Tac. Using immunofluorescence and immunoelectron microscopy, the COOH- terminal cytoplasmic domain of TGN38 was found to be sufficient for localization of the chimeric proteins to the TGN. Deletion analysis identified an 11-amino acid segment containing the critical sequence, YQRL, as being sufficient for TGN localization. TGN localization was abrogated by mutation of the tyrosine or leucine residues in this sequence to alanine, or of the arginine residue to aspartate. In addition to specifying TGN localization, the 11-amino acid segment was active as an internalization signal, although the property of internalization alone was insufficient to confer TGN localization. Overexpression of chimeric proteins containing TGN localization determinants resulted in their detection at the plasma membrane and in intracellular vesicles, and abolished detection of endogenous TGN38. These results suggest that discrete cytoplasmic determinants can mediate protein localization to the TGN, and reveal a novel role for tyrosine-based motifs in this process.     

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.     

A family of proteins with gamma-adaptin and VHS domains that facilitate trafficking between the trans-Golgi network and the vacuole/lysosome

We have cloned and characterized members of a novel family of proteins, the GGAs. These proteins contain an NH(2)-terminal VHS domain, one or two coiled-coil domains, and a COOH-terminal domain homologous to the COOH-terminal "ear" domain of gamma-adaptin. However, unlike gamma-adaptin, the GGAs are not associated with clathrin-coated vesicles or with any of the components of the AP-1 complex. GGA1 and GGA2 are also not associated with each other, although they colocalize on perinuclear membranes. Immunogold EM shows that these membranes correspond to trans elements of the Golgi stack and the TGN. GST pulldown experiments indicate that the GGA COOH-terminal domains bind to a subset of the proteins that bind to the gamma-adaptin COOH-terminal domain. In yeast there are two GGA genes. Deleting both of these genes results in missorting of the vacuolar enzyme carboxypeptidase Y, and the cells also have a defective vacuolar morphology phenotype. These results indicate that the function of the GGAs is to facilitate the trafficking of proteins between the TGN and the vacuole, or its mammalian equivalent, the lysosome.     

A tyrosine-based motif and a casein kinase II phosphorylation site regulate the intracellular trafficking of the varicella-zoster virus glycoprotein I, a protein localized in the trans-Golgi network.

We have studied the intracellular trafficking of the envelope glycoprotein I (gpI) of the varicella-zoster virus, a human herpes virus whose assembly is believed to occur in the trans-Golgi network (TGN) and/or in endocytic compartments. When expressed in HeLa cells in the absence of additional virally encoded factors, this type-I membrane protein localizes to the TGN and cycles between this compartment and the cell surface. The expression of gpI promotes the recruitment of the AP-1 Golgi-specific assembly proteins onto TGN membranes, strongly suggesting that gpI, like the mannose 6-phosphate receptors, can leave the TGN in clathrin-coated vesicles for subsequent transport to endosomes. Its return from the cell surface to the TGN also occurs through endosomes. The transfer of the gpI cytoplasmic domain onto a reporter molecule shows that this domain is sufficient to confer TGN localization. Mutational analysis of this domain indicates that proper subcellular localization and cycling of gpI depend on two different determinants, a tyrosine-containing tetrapeptide related to endocytosis sorting signals and a cluster of acidic amino acids containing casein kinase II phosphorylatable residues. Thus, the VZV gpI and the mannose 6-phosphate receptors, albeit localized in different intracellular compartments at steady-state, follow similar trafficking pathways and share similar sorting mechanisms.     

Differential distribution of low-density lipoprotein-receptor-related protein (LRP) and megalin in polarized epithelial cells is determined by their cytoplasmic domains

Megalin and the low-density lipoprotein (LDL) receptor-related protein (LRP) are two large members of the LDL receptor family that bind and endocytose multiple ligands. The molecular and cellular determinants that dictate the sorting behavior of these receptors in polarized epithelial cells are largely unknown. Megalin is found apically distributed, whereas the limited information on LRP indicates its polarity. We show here that in Madin-Darby canine kidney cells, both endogenous LRP and a minireceptor containing the fourth ligand-binding, transmembrane and LRP cytosolic domains were basolaterally sorted. In contrast, minireceptors that either lacked the cytoplasmic domain or had the tyrosine in the NPTY motif mutated to alanine showed a preferential apical distribution. In LLC-PK1 cells, endogenous megalin was found exclusively in the apical membrane. Studies were also done using chimeric proteins harboring the cytosolic tail of megalin, one with the fourth ligand-binding domain of LRP and the other two containing the green fluorescent protein as the ectodomain and transmembrane domains of either megalin or LRP. Findings from these experiments showed that the cytosolic domain of megalin is sufficient for apical sorting, and that the megalin transmembrane domain promotes association with lipid rafts. In conclusion, we show that LRP and megalin both contain sorting information in their cytosolic domains that directs opposite polarity, basolateral for LRP and apical for megalin. Additionally, we show that the NPTY motif in LRP is important for basolateral sorting and the megalin transmembrane domain directs association with lipid rafts .     

SNX1 defines an early endosomal recycling exit for sortilin and mannose 6-phosphate receptors

Mannose-6-phosphate receptors (MPRs) transport lysosomal hydrolases from the trans Golgi network (TGN) to endosomes. Recently, the multi-ligand receptor sortilin has also been implicated in this transport, but the transport carriers involved herein have not been identified. By quantitative immuno-electron microscopy, we localized endogenous sortilin of HepG2 cells predominantly to the TGN and endosomes. In the TGN, sortilin colocalized with MPRs in the same clathrin-coated vesicles. In endosomes, sortilin and MPRs concentrated in sorting nexin 1 (SNX1)-positive buds and vesicles. SNX1 depletion by small interfering RNA resulted in decreased pools of sortilin in the TGN and an increase in lysosomal degradation. These data indicate that sortilin and MPRs recycle to the TGN in SNX1-dependent carriers, which we named endosome-to-TGN transport carriers (ETCs). Notably, ETCs emerge from early endosomes (EE), lack recycling plasma membrane proteins and by three-dimensional electron tomography exhibit unique structural features. Hence, ETCs are distinct from hitherto described EE-derived membranes involved in recycling. Our data emphasize an important role of EEs in recycling to the TGN and indicate that different, specialized exit events occur on the same EE vacuole.     

Targeting of glycoprotein I (gE) of varicella-zoster virus to the trans-Golgi network by an AYRV sequence and an acidic amino acid-rich patch in the cytosolic domain of the molecule.

Previous studies suggested that varicella-zoster virus (VZV) envelope glycoproteins (gps) are selectively transported to the trans-Golgi network (TGN) and that the cytosolic domain of gpI (gE) targets it to the TGN. To identify targeting signals in the gpI cytosolic domain, intracellular protein trafficking was studied in transfected cells expressing chimeric proteins in which a full-length or mutated gpI cytosolic domain was fused to the gpI transmembrane domain and interleukin-2 receptor (tac) ectodomain. Expressed protein was visualized with antibodies to tac. A targeting sequence (AYRV) and a second, acidic amino acid-rich region of the gpI cytosolic domain (putative signal patch) were each sufficient to cause expressed protein to colocalize with TGN markers. This targeting was lost when the tyrosine of the AYRV sequence was replaced with glycine or lysine, when arginine was replaced with glutamic acid, or when valine was substituted with lysine. In contrast, tyrosine could be replaced by phenylalanine and valine could be substituted with leucine. Mutation of alanine to aspartic acid or deletion of alanine abolished TGN targeting. Exposure of transfected cells to antibodies to the tac ectodomain revealed that the TCN targeting of expressed tac-gpI chimeric proteins occurred as a result of selective retrieval from the plasmalemma. These data suggest that the AYRV sequence and a second signaling patch in the cytosolic domain of gpI are responsible for its targeting to the TGN. The observations also support the hypothesis that the TGN plays a critical role in the envelopment of VZV.     

Memapsin 2 (beta-secretase) cytosolic domain binds to the VHS domains of GGA1 and GGA2: implications on the endocytosis mechanism of memapsin 2

Memapsin 2, or beta-secretase, is a membrane-anchored aspartic protease that initiates the cleavage of beta-amyloid precursor protein (APP) leading to the production of beta-amyloid peptide in the brain and the onset of Alzheimer's disease. Memapsin 2 and APP are both endocytosed into endosomes for cleavage. Here we show that the cytosolic domain of memapsin 2, but not that of memapsin 1, binds the VHS domains of GGA1 and GGA2. Gel-immobilized VHS domains of GGA1 and GGA2 also bound to full-length memapsin 2 from cell mammalian lysates. Mutagenesis studies established that Asp(496), Leu(499) and Leu(500) were essential for the binding. The spacing of these three residues in memapsin 2 is identical to those in the cytosolic domains of mannose-6-phosphate receptors, sortilin and low density lipoprotein receptor-related protein 3. These observations suggest that the endocytosis and intracellular transport of memapsin 2, mediated by its cytosolic domain, may involve the binding of GGA1 and GGA2.     

A family of ADP-ribosylation factor effectors that can alter membrane transport through the trans-Golgi

A family of three structurally related proteins were cloned from human cDNA libraries by their ability to interact preferentially with the activated form of human ADP-ribosylation factor 3 (ARF3) in two-hybrid assays. The specific and GTP-dependent binding was later confirmed through direct protein binding of recombinant proteins. The three proteins share large ( approximately 300 residues) domains at their N termini that are 60-70% identical to each other and a shorter (73 residues) domain at their C termini with 70% homology to the C-terminal "ear" domain of gamma-adaptin. Although GGA1 is found predominantly as a soluble protein by cell fractionation, all three proteins were found to localize to the trans-Golgi network (TGN) by indirect immunofluorescence. The binding of GGAs to TGN was sensitive to brefeldin A, consistent with this being an ARF-dependent event. Thus, these proteins have been named Golgi-localizing, gamma-adaptin ear homology domain, ARF-binding proteins, or GGAs. The finding that overexpression of GGAs was sufficient to alter the distribution of markers of the TGN (TGN38 and mannose 6-phosphate receptors) led us to propose that GGAs are effectors for ARFs that function in the regulation of membrane traffic through the TGN.