Analysis of the carboxypeptidase D cytoplasmic domain: Implications in intracellular trafficking

Metallocarboxypeptidase D (CPD) is a type 1 transmembrane protein that functions in the processing of proteins that transit the secretory pathway. Previously, CPD was found to be enriched in the trans Golgi network (TGN) and to cycle between this compartment and the cell surface. In the present study, the roles of specific regions of the CPD cytosolic tail in intracellular trafficking were investigated in the AtT-20 cell line. When the CPD transmembrane region and cytosolic tail are attached to the C-terminus of albumin, this protein is retained in the TGN and cycles to the cell surface. Deletion analysis indicates that a C-terminal region functions in TGN-retention; removal of 10 amino acids from the C-terminus greatly increases the amount of fusion protein that enters nascent vesicles, which bud from the Golgi, but does not affect the half-life of the fusion protein or the ability of cell surface protein to return to the TGN. Because the 10-residue deletion disrupts a casein kinase 2 (CK2) consensus site, the two Thr in this site (TDT) were mutated to either Ala (ADA) or Glu (EDE). Neither mutation has an increased rate of budding from the TGN, although the ADA mutant has a shorter half-life than either the wild type sequence or the EDE mutant. Adaptor protein-1 and -2 bind to most of the deletion mutants, the EDE point mutant, and the CK2-phosphorylated CPD tail, but not to the wild type tail. Taken together, these results suggest that CPD localization to the TGN requires both static retention involving the C-terminal domain and phosphorylation at a CK2 site, which regulates the binding of adaptor proteins.     

Two Separate Signals Act Independently to Localize a Yeast Late Golgi Membrane Protein through a Combination of Retrieval and Retention

The localization of proteins to late-Golgi membranes (TGN) of Saccharomyces cerevisiae is conferred by targeting motifs containing aromatic residues in the cytosolic domains of these proteins. These signals could act by directing retrieval from a post-Golgi compartment or by preventing exit from the TGN. To investigate the mechanism of localization of yeast TGN proteins, we used the heterologous protein A-ALP (consisting of the cytosolic domain of dipeptidyl aminopeptidase A [DPAP A] fused to the transmembrane and luminal domains of the vacuolar protein alkaline phosphatase [ALP]), which localizes to the yeast TGN. Insertion of the aromatic residue–based TGN localization motif (FXFXD) of DPAP A into the cytosolic domain of ALP results in a protein that resides in the TGN. We demonstrate that the FXFXD motif confers Golgi localization through retrieval from a post-Golgi compartment by detecting a post-Golgi processed form of this protein in the TGN. We present an assay that uncouples retrieval-mediated Golgi localization from static retention-based localization, allowing measurement of the rate at which proteins exit the yeast TGN. We also demonstrate that the cytosolic domain of DPAP A contains additional information, separate from the retrieval motif, that slows exit from the TGN. We propose a model for DPAP A localization that involves two distinct mechanisms: one in which the FXFXD motif directs retrieval from a post-Golgi compartment, and a second that slows the rate at which DPAP A exits the TGN.     

OsGAP1 functions as a positive regulator of OsRab11-mediated TGN to PM or vacuole trafficking

The Ypt/Rab family of small G-proteins is important in regulating vesicular transport. Rabs hydrolyze GTP very slowly on their own and require GTPase-activating proteins (GAPs). Here we report the identification and characterization of OsGAP1, a Rab-specific rice GAP. OsGAP1 strongly stimulated OsRab8a and OsRab11, which are homologs of the mammalian Rab8 and Rab11 proteins that are essential for Golgi to plasma membrane (PM) and trans-Golgi network (TGN) to PM trafficking, respectively. Substitution of two invariant arginines within the catalytic domain of Oryza sativa GTPase-activating protein 1 (OsGAP1) with alanines significantly inhibited its GAP activity. In vivo targeting experiments revealed that OsGAP1 localizes to the TGN or pre-vacuolar compartment (PVC). A yeast expression system demonstrated that wild-type OsGAP1 facilitates O. sativa dissociation inhibitor 3 (OsGDI3)-catalyzed OsRab11 recycling at an early stage, but the OsGAP1(R385A) and (R450A) mutants do not. Thus, GTP hydrolysis is essential for Rab recycling. Moreover, expression of the OsGAP1 mutants in Arabidopsis protoplasts inhibited the trafficking of some cargo proteins, including the PM-localizing H+-ATPase-green fluorescent protein (GFP) and Ca2+-ATPase8-GFP and the central vacuole-localizing Arabidopsis aleurain-like protein (AALP)-GFP. The OsGAP1 mutants caused these proteins to accumulate at the Golgi apparatus. Surprisingly, OsRab11 overproduction relieved the inhibitory effect of the OsGAP1 mutants on vesicular trafficking. OsRab8a had no such effect. Thus, the OsGAP1 mutants may inhibit TGN to PM or central vacuole trafficking because they induce the sequestration of endogenous Rab11. We propose that OsGAP1 facilitates vesicular trafficking from the TGN to the PM or central vacuole by both stimulating the GTPase activity of OsRab11 and increasing the recycling of inactive OsRab11.     

Systematic analysis of SNARE molecules in Arabidopsis: dissection of the post-Golgi network in plant cells

In all eucaryotic cells, specific vesicle fusion during vesicular transport is mediated by membrane-associated proteins called SNAREs (soluble N-ethyl-maleimide sensitive factor attachment protein receptors). Sequence analysis identified a total of 54 SNARE genes (18 Qa-SNAREs/Syntaxins, 11 Qb-SNAREs, 8 Qc-SNAREs, 14 R-SNAREs/VAMPs and 3 SNAP-25) in the Arabidopsis genome. Almost all of them were ubiquitously expressed through out all tissues examined. A series of transient expression assays using green fluorescent protein (GFP) fused proteins revealed that most of the SNARE proteins were located on specific intracellular compartments: 6 in the endoplasmic reticulum, 9 in the Golgi apparatus, 4 in the trans-Golgi network (TGN), 2 in endosomes, 17 on the plasma membrane, 7 in both the prevacuolar compartment (PVC) and vacuoles, 2 in TGN/PVC/vacuoles, and 1 in TGN/PVC/plasma membrane. Some SNARE proteins showed multiple localization patterns in two or more different organelles, suggesting that these SNAREs shuttle between the organelles. Furthermore, the SYP41/SYP61-residing compartment, which was defined as the TGN, was not always located along with the Golgi apparatus, suggesting that this compartment is an independent organelle distinct from the Golgi apparatus. We propose possible combinations of SNARE proteins on all subcellular compartments, and suggest the complexity of the post-Golgi membrane traffic in higher plant cells.     

Plant TGNs: dynamics and physiological functions

In all eukaryotic cells, a membrane trafficking system connects the post-Golgi organelles, including the trans-Golgi network (TGN), endosomes, and vacuoles. This complex network plays critical roles in several higher-order functions in multicellular organisms. The TGN, one of the important organelles for protein transport in the post-Golgi network, functions as a sorting station, where cargo proteins are directed to the appropriate post-Golgi compartments. The TGN has been considered to be a compartment belonging to the Golgi apparatus, located on the trans side of the Golgi apparatus. However, in plant cells, recent studies have suggested that the TGN is an independent, dynamic organelle that possesses features different than those of TGNs in animal and yeast cells. In this review, we summarize recent progress regarding the dynamics and physiological functions of the plant TGN.     

The v-sis oncoprotein loses transforming activity when targeted to the early Golgi complex

The location of autocrine interactions between the v-sis protein and PDGF receptors remains uncertain and controversial. To examine whether receptor-ligand interactions can occur intracellularly, we have constructed fusion proteins that anchor v-sis to specific intracellular membranes. Fusion of a cis-Golgi retention signal from a coronavirus E1 glycoprotein to v-sis protein completely abolished its transforming ability when transfected into NIH3T3 cells. Fusion proteins incorporating mutations in this retention signal were not retained within the Golgi complex but instead were transported to the cell surface, resulting in efficient transformation. All chimeric proteins were shown to dimerize properly. Derivatives of some of these constructs were also constructed bearing the cytoplasmic tail from the glycoprotein of vesicular stomatitis virus (VSV-G). These constructs allowed examination of subcellular localization by double-label immunofluorescence, using antibodies that distinguish between the extracellular PDGF-related domain and the VSV-G cytoplasmic tail. Colocalization of sis-E1-G with Golgi markers confirmed its targeting to the early Golgi complex. The sis-E1 constructs, targeted to the early Golgi complex, exhibited no proteolytic processing whereas the mutant forms of sis-E1 exhibited normal proteolytic processing. Treatment with suramin, a polyanionic compound that disrupts ligand/receptor interactions at the cell surface, was able to revert the transformed phenotype induced by the mutant sis-E1 constructs described here. Our results demonstrate that autocrine interactions between the v-sis oncoprotein and PDGF receptors within the early Golgi complex do not result in functional signal transduction. Another v-sis fusion protein was constructed by attaching the transmembrane domain and COOH-terminus of TGN38, a protein that localizes to the trans-Golgi network (TGN). This construct was primarily retained intracellularly, although some of the fusion protein reached the surface. Deletion of the COOH-terminal region of the TGN38 retention signal abrogated the TGN-localization, as evidenced by very prominent cell surface localization, and resulted in increased transforming activity. The behavior of the sis-TGN38 derivatives is discussed within the context of the properties of TGN38 itself, which is known to recycle from the cell surface to the TGN.     

Synthetic genetic interactions with temperature-sensitive clathrin in Saccharomyces cerevisiae. Roles for synaptojanin-like Inp53p and dynamin-related Vps1p in clathrin-dependent protein sorting at the trans-Golgi network

Clathrin is involved in selective protein transport at the Golgi apparatus and the plasma membrane. To further understand the molecular mechanisms underlying clathrin-mediated protein transport pathways, we initiated a genetic screen for mutations that display synthetic growth defects when combined with a temperature-sensitive allele of the clathrin heavy chain gene (chc1-521) in Saccharomyces cerevisiae. Mutations, when present in cells with wild-type clathrin, were analyzed for effects on mating pheromone alpha-factor precursor maturation and sorting of the vacuolar protein carboxypeptidase Y as measures of protein sorting at the yeast trans-Golgi network (TGN) compartment. By these criteria, two classes of mutants were obtained, those with and those without defects in protein sorting at the TGN. One mutant with unaltered protein sorting at the TGN contains a mutation in PTC1, a type 2c serine/threonine phosphatase with widespread influences. The collection of mutants displaying TGN sorting defects includes members with mutations in previously identified vacuolar protein sorting genes (VPS), including the dynamin family member VPS1. Striking genetic interactions were observed by combining temperature-sensitive alleles of CHC1 and VPS1, supporting the model that Vps1p is involved in clathrin-mediated vesicle formation at the TGN. Also in the spectrum of mutants with TGN sorting defects are isolates with mutations in the following: RIC1, encoding a product originally proposed to participate in ribosome biogenesis; LUV1, encoding a product potentially involved in vacuole and microtubule organization; and INP53, encoding a synaptojanin-like inositol polyphosphate 5-phosphatase. Disruption of INP53, but not the related INP51 and INP52 genes, resulted in alpha-factor maturation defects and exacerbated alpha-factor maturation defects when combined with chc1-521. Our findings implicate a wide variety of proteins in clathrin-dependent processes and provide evidence for the selective involvement of Inp53p in clathrin-mediated protein sorting at the TGN.     

Receptor-mediated transport of vacuolar proteins: a critical analysis and a new model

In this article we challenge the widely accepted view that receptors for soluble vacuolar proteins (VSRs) bind to their ligands at the trans-Golgi network (TGN) and transport this cargo via clathrin-coated vesicles (CCV) to a multivesicular prevacuolar compartment. This notion, which we term the “classical model” for vacuolar protein sorting, further assumes that low pH in the prevacuolar compartment causes VSR–ligand dissociation, resulting in a retromer-mediated retrieval of the VSRs to the TGN. We have carefully evaluated the literature with respect to morphology and function of the compartments involved, localization of key components of the sorting machinery, and conclude that there is little direct evidence in its favour. Firstly, unlike mammalian cells where the sorting receptor for lysosomal hydrolases recognizes its ligand in the TGN, the available data suggests that in plants VSRs interact with vacuolar cargo ligands already in the endoplasmic reticulum. Secondly, the evidence supporting the packaging of VSR–ligand complexes into CCV at the TGN is not conclusive. Thirdly, the prevacuolar compartment appears to have a pH unsuitable for VSR–ligand dissociation and lacks the retromer core and the sorting nexins needed for VSR recycling. We present an alternative model for protein sorting in the TGN that draws attention to the much overlooked role of Ca²⁺ in VSR–ligand interactions and which may possibly also be a factor in the sequestration of secretory proteins.     

Isolation of a fraction enriched in the trans-Golgi network from baby hamster kidney cells

Incubation of cultured cells at 20 degrees C blocks the transport of newly synthesized plasma membrane proteins, and the proteins accumulate intracellularly in a terminally glycosylated form. When baby hamster kidney cells are infected with the ts O45 mutant of vesicular stomatitis virus, and incubated at 20 degrees C, the terminally glycosylated spike glycoprotein G of the virus accumulates in the membranes of a tubular network localized on the trans side of the Golgi cisternae, the trans-Golgi network (TGN). We have used the G protein of ts O45 as a marker for the TGN and isolated a TGN fraction using a combination of conventional cell fractionation techniques and immunoisolation. The TGN was separated from the bulk of the endoplasmic reticulum, mitochondria, lysosomes, plasma membrane, and endosomes, while the activity of trans-Golgi marker galactosyltransferase copurified with the G protein. Using G protein as the TGN marker we have determined that the TGN was enriched 25-fold in the final fraction relative to the total homogenate. Several polypeptides (Mr 75,000, 87,000, 92,000, and 120,000) copurified with the G protein in the isolated TGN fraction and most likely represent resident markers of the compartment.     

The functioning of the yeast Golgi apparatus requires an ER protein encoded by ANP1, a member of a new family of genes affecting the secretory pathway.

Mnt1p is an alpha 1.2-mannosyltransferase which resides in an early compartment of the Saccharomyces cerevisiae Golgi apparatus. We have shown that the signal-anchor region is sufficient, and the transmembrane domain necessary, for its normal Golgi localization. This is similar to the transmembrane domain-mediated retention of mammalian glycosyltransferases, and distinct from the tail-mediated recycling retention of certain mammalian and yeast trans-Golgi proteins. To examine the mechanism involved in transmembrane domain-mediated retention, we have isolated six classes of mutants which fail to retain Mnt1p-reporter fusions in the early Golgi. These mutants all show additional phenotypes which are consistent with alterations in Golgi function. We have called the mutant classes 'gem', for Golgi enzyme maintenance. GEM3 is identical to the previously cloned gene ANP1, and homologous to VAN1 and MNN9. Together, these define a new class of proteins involved in the organization and functioning of the secretory pathway. Interestingly, Anp1p is localized to the endoplasmic reticulum (ER), implying that some function of the ER is required to maintain a functional Golgi apparatus.     

Distinct domains within Vps35p mediate the retrieval of two different cargo proteins from the yeast prevacuolar/endosomal compartment

Resident membrane proteins of the trans-Golgi network (TGN) of Saccharomyces cerevisiae are selectively retrieved from a prevacuolar/late endosomal compartment. Proper cycling of the carboxypeptidase Y receptor Vps10p between the TGN and prevacuolar compartment depends on Vps35p, a hydrophilic peripheral membrane protein. In this study we use a temperature-sensitive vps35 allele to show that loss of Vps35p function rapidly leads to mislocalization of A-ALP, a model TGN membrane protein, to the vacuole. Vps35p is required for the prevacuolar compartment-to-TGN transport of both A-ALP and Vps10p. This was demonstrated by phenotypic analysis of vps35 mutant strains expressing A-ALP mutants lacking either the retrieval or static retention signals and by an assay for prevacuolar compartment-to-TGN transport. A novel vps35 allele was identified that was defective for retrieval of A-ALP but functional for retrieval of Vps10p. Moreover, several other vps35 alleles were identified with the opposite characteristics: they were defective for Vps10p retrieval but near normal for A-ALP localization. These data suggest a model in which distinct structural features within Vps35p are required for associating with the cytosolic domains of each cargo protein during the retrieval process.     

The dynamic nature of the Golgi complex

The intracellular transport of newly synthesized G protein of vesicular stomatitis virus is blocked at 20 degrees C and this spanning membrane glycoprotein accumulates in the last Golgi compartment, the trans Golgi-network (TGN). Previous morphological evidence suggested that the TGN enlarged significantly under this condition. In the present study we have used stereological procedures to estimate the volume and surface area of the Golgi stack and the TGN of baby hamster kidney cells under different conditions. The results indicate that the increase in the size of the TGN at 20 degrees C is accompanied by a significant decrease in the surface area and volume of the preceding Golgi compartments. A similar effect is also seen in uninfected cells at 20 degrees C, as well as during normal (37 degrees C) infection with Semliki Forest virus. In the latter case, however, the decrease in the size of the Golgi stack and the increase in that of the TGN is not accompanied by inhibition of transport from the Golgi complex to the cell surface. The results indicate that the Golgi stack and the TGN are dynamic and interrelated structures that are capable of rapid alteration in total surface area in response to changes in the rates of membrane transport.     

A novel mechanism for localizing membrane proteins to yeast trans-Golgi network requires function of synaptojanin-like protein

Localization of resident membrane proteins to the yeast trans-Golgi network (TGN) involves both their retrieval from a prevacuolar/endosomal compartment (PVC) and a "slow delivery" mechanism that inhibits their TGN-to-PVC transport. A screen for genes required for the slow delivery mechanism uncovered INP53, a gene encoding a phosphoinositide phosphatase. A retrieval-defective model TGN protein, A(F-->A)-ALP, was transported to the vacuole in inp53 mutants approximately threefold faster than in wild type. Inp53p appears to function in a process distinct from PVC retrieval because combining inp53 with mutations that block retrieval resulted in a much stronger phenotype than either mutation alone. In vps27 strains defective for both anterograde and retrograde transport out of the PVC, a loss of Inp53p function markedly accelerated the rate of transport of TGN residents A-ALP and Kex2p into the PVC. Inp53p function is cargo specific because a loss of Inp53p function had no effect on the rate of Vps10p transport to the PVC in vps27 cells. The rate of early secretory pathway transport appeared to be unaffected in inp53 mutants. Cell fractionation experiments suggested that Inp53p associates with Golgi or endosomal membranes. Taken together, these results suggest that a phosphoinositide signaling event regulates TGN-to-PVC transport of select cargo proteins.     

BEX5/RabA1b Regulates trans-Golgi Network-to-Plasma Membrane Protein Trafficking in Arabidopsis

Constitutive endocytic recycling is a crucial mechanism allowing regulation of the activity of proteins at the plasma membrane and for rapid changes in their localization, as demonstrated in plants for PIN-FORMED (PIN) proteins, the auxin transporters. To identify novel molecular components of endocytic recycling, mainly exocytosis, we designed a PIN1-green fluorescent protein fluorescence imaging-based forward genetic screen for Arabidopsis thaliana mutants that showed increased intracellular accumulation of cargos in response to the trafficking inhibitor brefeldin A (BFA). We identified bex5 (for BFA-visualized exocytic trafficking defective), a novel dominant mutant carrying a missense mutation that disrupts a conserved sequence motif of the small GTPase, RAS GENES FROM RAT BRAINA1b. bex5 displays defects such as enhanced protein accumulation in abnormal BFA compartments, aberrant endosomes, and defective exocytosis and transcytosis. BEX5/RabA1b localizes to trans-Golgi network/early endosomes (TGN/EE) and acts on distinct trafficking processes like those regulated by GTP exchange factors on ADP-ribosylation factors GNOM-LIKE1 and HOPM INTERACTOR7/BFA-VISUALIZED ENDOCYTIC TRAFFICKING DEFECTIVE1, which regulate trafficking at the Golgi apparatus and TGN/EE, respectively. All together, this study identifies Arabidopsis BEX5/RabA1b as a novel regulator of protein trafficking from a TGN/EE compartment to the plasma membrane.     

Asymmetric CLASP-dependent nucleation of noncentrosomal microtubules at the trans-Golgi network

Proper organization of microtubule arrays is essential for intracellular trafficking and cell motility. It is generally assumed that most if not all microtubules in vertebrate somatic cells are formed by the centrosome. Here we demonstrate that a large number of microtubules in untreated human cells originate from the Golgi apparatus in a centrosome-independent manner. Both centrosomal and Golgi-emanating microtubules need gamma-tubulin for nucleation. Additionally, formation of microtubules at the Golgi requires CLASPs, microtubule-binding proteins that selectively coat noncentrosomal microtubule seeds. We show that CLASPs are recruited to the trans-Golgi network (TGN) at the Golgi periphery by the TGN protein GCC185. In sharp contrast to radial centrosomal arrays, microtubules nucleated at the peripheral Golgi compartment are preferentially oriented toward the leading edge in motile cells. We propose that Golgi-emanating microtubules contribute to the asymmetric microtubule networks in polarized cells and support diverse processes including post-Golgi transport to the cell front.     

Localization of the Lys, Asp, Glu, Leu tetrapeptide receptor to the Golgi complex and the intermediate compartment in mammalian cells

The carboxyl-terminal Lys-Asp-Glu-Leu (KDEL), or a closely-related sequence, is important for ER localization of both lumenal as well as type II membrane proteins. This sequence functions as a retrieval signal at post-ER compartment(s), but the exact compartment(s) where the retrieval occurs remains unresolved. With an affinity-purified antibody against the carboxyl-terminal sequence of the mammalian KDEL receptor, we have investigated its subcellular localization using immunogold labeling on thawed cryosections of different tissues, such as mouse spermatids and rat pancreas, as well as HeLa, Vero, NRK, and mouse L cells. We show that rab1 is an excellent marker of the intermediate compartment, and we use this marker, as well as budding profiles of the mouse hepatitis virus (MHV) in cells infected with this virus, to identify this compartment. Our results demonstrate that the KDEL receptor is concentrated in the intermediate compartment, as well as in the Golgi stack. Lower but significant labeling was detected in the rough ER. In general, only small amounts of the receptor were detected on the trans side of the Golgi stack, including the trans- Golgi network (TGN) of normal cells and tissues. However, some stress conditions, such as infection with vaccinia virus or vesicular stomatitis virus, as well as 20 degrees C or 43 degrees C treatment, resulted in a significant shift of the distribution towards the trans- TGN side of the Golgi stack. This shift could be quantified in HeLa cells stably expressing a TGN marker. No significant labeling was detected in structures distal to the TGN under all conditions tested. After GTP gamma S treatment of permeabilized cells, the receptor was detected in the beta-COP-containing buds/vesicles that accumulate after this treatment, suggesting that these vesicles may transport the receptor between compartments. We propose that retrieval of KDEL- containing proteins occurs at multiple post-ER compartments up to the TGN along the exocytotic pathway, and that within this pathway, the amounts of the receptor in different compartments varies according to physiological conditions.     

Localization of a yeast early Golgi mannosyltransferase, Och1p, involves retrograde transport

To analyze the mechanism of integral membrane protein localization in the early Golgi apparatus of Saccharomyces cerevisiae, we have used Och1p, a cis-Golgi mannosyltransferase. A series of influenza virus hemagglutinin (HA) epitope-tagged fusion proteins was constructed in which invertase is appended to the Golgi-luminal carboxy terminus of full-length Och1p. Several constructs included a Kex2p cleavage site between the Och1p and invertase moieties to monitor transit to the Kex2p-containing TGN. Cells expressing an Och1p-invertase fusion do not secrete invertase, but those expressing an Och1p-Kex2p site-invertase fusion protein secrete high levels of invertase in a Kex2p-dependent manner. The Och1p-Kex2p site-invertase fusion protein is cleaved with a half-time of 5 min, and the process proceeds to completion. Before cleavage the protein receives glycosyl modifications indicative of passage through the medial- and trans-Golgi, therefore cleavage occurs after ordered anterograde transport through the Golgi to the TGN. Transit to distal compartments is not induced by the invertase moiety, since noninvertase fusion constructs encounter the same glycosyltransferases and Kex2p as well. The Och1p-HA moiety, irrespective of whether it is generated by cleavage of the fusion protein in the TGN or synthesized de novo, is degraded with a half-time of about 60 min. Thus, the half-time of degradation is 12-fold longer than the time required to reach the TGN. At steady state, de novo- synthesized and TGN-generated HA epitope-tagged Och1p reside in a compartment with a buoyant density identical to that of wild-type Och1p and distinct from that of the vacuole or the TGN. Finally, och1 null cells that express an Ochlp fusion construct known to rapidly encounter the TGN glycosylate invertase to the same extent as wild-type cells, indicating that they have phenotypically wild-type Och1p activity. These results lead us to propose a model for Och1p-HA localization that involves movement to distal compartments, at least as far as the TGN, followed by retrieval to the cis compartment, presumably by vesicular transport.     

Assembly,Target‐Signaling and Intracellular Transport of Tyrosinase Gene Family Proteins in the Initial Stage of Melanosome Biogenesis

Assembly, target‐signaling and transport of tyrosinase gene family proteins at the initial stage of melanosome biogenesis are reviewed based on our own discoveries. Melanosome biogenesis involves four stages of maturation with distinct morphological and biochemical characteristics that reflect distinct processes of the biosynthesis of structural and enzymatic proteins, subsequent structural organization and melanin deposition occurring in these particular cellular compartments. The melanosomes share many common biological properties with the lysosomes. The stage I melanosomes appear to be linked to the late endosomes. Most of melanosomal proteins are glycoproteins that should be folded or assembled correctly in the ER through interaction with calnexin, a chaperone associated with melanogenesis. These melanosomal glycoproteins are then accumulated in the trans Golgi network (TGN) and transported to the melanosomal compartment. During the formation of transport vesicles, coat proteins assemble on the cytoplasmic face of TGN to select their cargos by interacting directly or indirectly with melanosomal glycoproteins to be transported. Adapter protein‐3 (AP‐3) is important for intracellular transport of tyrosinase gene family proteins from TGN to melanosomes. Tyrosinase gene family proteins possess a di‐leucine motif in their cytoplasmic tail, to which AP‐3 appears to bind. Thus, the initial cascade of melanosome biogenesis is regulated by several factors including: 1) glycosylation of tyrosinase gene family proteins and their correct folding and assembly within ER and Golgi, and 2) supply of specific signals necessary for intracellular transport of these glycoproteins by vesicles from Golgi to melanosomes.     

Targeting of proteins to the Golgi apparatus

 The proteins that reside in the Golgi carry out functions associated with post-translational modifications, including glycosylation and proteolytic processing, membrane transport, recycling of endoplasmic reticulum proteins and maintenance of the structural organisation of the organelle itself. The latter includes Golgi stacking, interconnections between stacks and the microtubule-dependent positioning of the organelle within the cell. There are a number of distinct groups of Golgi membrane proteins, including glycosyltransferases, recycling trans-Golgi network (TGN) proteins, peripheral membrane proteins and receptors. Considerable effort has been directed at understanding the basis of the localisation of Golgi glycosyltransferases and recycling TGN proteins; in both cases there is increasing evidence that multiple signals may be involved in their specific localisation. A number of models for the Golgi retention of glycosyltransferases have been proposed including oligomerisation, lipid-mediated sorting and intra-Golgi retrograde transport. More information is required to determine the contribution of each of these potential mechanisms in the targeting of different glycosyltransferases. Future work is also likely to focus on the relationship between the localisation of resident Golgi proteins and the maintenance of Golgi structure. Accepted: 15 October 1997     

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.