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1.
Ca2+ import into the lumen of the trans-Golgi network (TGN) by the secretory pathway calcium ATPase1 (SPCA1) is required for the sorting of secretory cargo. How is Ca2+ retained in the lumen of the Golgi, and what is its role in cargo sorting? We show here that a soluble, lumenal Golgi resident protein, Cab45, is required for SPCA1-dependent Ca2+ import into the TGN; it binds secretory cargo in a Ca2+-dependent reaction and is required for its sorting at the TGN.  相似文献   

2.
Knockdown of the actin-severing protein actin-depolymerizing factor (ADF)/cofilin inhibited export of an exogenously expressed soluble secretory protein from Golgi membranes in Drosophila melanogaster and mammalian tissue culture cells. A stable isotope labeling by amino acids in cell culture mass spectrometry–based protein profiling revealed that a large number of endogenous secretory proteins in mammalian cells were not secreted upon ADF/cofilin knockdown. Although many secretory proteins were retained, a Golgi-resident protein and a lysosomal hydrolase were aberrantly secreted upon ADF/cofilin knockdown. Overall, our findings indicate that inactivation of ADF/cofilin perturbed the sorting of a subset of both soluble and integral membrane proteins at the trans-Golgi network (TGN). We suggest that ADF/cofilin-dependent actin trimming generates a sorting domain at the TGN, which filters secretory cargo for export, and that uncontrolled growth of this domain causes missorting of proteins. This type of actin-dependent compartmentalization and filtering of secretory cargo at the TGN by ADF/cofilin could explain sorting of proteins that are destined to the cell surface.  相似文献   

3.
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.  相似文献   

4.
Highlights? ADF/cofilin localizes to the TGN and binds SPCA1 in an actin-dependent manner ? Dynamic actin regulates the activity of the Ca2+ pump SPCA1 ? ADF/cofilin facilitates secretory cargo sorting in a Ca2+-dependent manner  相似文献   

5.
M Seeger  G S Payne 《The EMBO journal》1992,11(8):2811-2818
We have investigated the role of clathrin in vacuolar protein sorting using yeast strains harboring a temperature-sensitive allele of clathrin heavy chain (chc1-ts). After a 5 min incubation at the non-permissive temperature (37 degrees C), the chc1-ts strains displayed a severe defect in the sorting of lumenal vacuolar proteins. Sorting of a vacuolar membrane protein, alkaline phosphatase, and transport to the surface of a cell wall protein, was not affected at 37 degrees C. In chc1-ts cells incubated at 37 degrees C, secretion of the missorted lumenal vacuolar protein carboxypeptidase Y (CPY) was blocked by the sec1 mutation which prevents fusion of secretory vesicles to the plasma membrane. Unexpectedly, chc1-ts cells incubated for extended periods at 37 degrees C regained the ability to sort CPY. Cells carrying deletions of the CHC1 gene (chc1 delta) also sorted CPY to the vacuole even when subjected to temperature shifts. Vacuolar delivery of CPY in chc1 delta cells was not blocked by sec1 suggesting that transport does not occur by secretion and endocytosis. These results provide in vivo evidence that clathrin plays a role in the Golgi complex in sorting of vacuolar proteins from the secretory pathway. With time, however, yeast cells lacking functional clathrin heavy chains are able to adapt in a way that allows restoration of vacuolar protein sorting in the Golgi complex. These conclusions clarify previous studies of chc1 delta cells which raised the possibility that clathrin is not involved in vacuolar protein sorting.  相似文献   

6.
《The Journal of cell biology》1993,121(6):1245-1256
We are studying intercompartmental protein transport to the yeast lysosome-like vacuole with a reconstitution assay using permeabilized spheroplasts that measures, in an ATP and cytosol dependent reaction, vacuolar delivery and proteolytic maturation of the Golgi-modified precursor forms of vacuolar hydrolases like carboxypeptidase Y (CPY). To identify the potential donor compartment in this assay, we used subcellular fractionation procedures that have uncovered a novel membrane-enclosed prevacuolar transport intermediate. Differential centrifugation was used to separate permeabilized spheroplasts into 15K and 150K g membrane pellets. Centrifugation of these pellets to equilibrium on sucrose density gradients separated vacuolar and Golgi complex marker enzymes into light and dense fractions, respectively. When the Golgi-modified precursor form of CPY (p2CPY) was examined (after a 5-min pulse, 30-s chase), as much as 30-40% fractionated with an intermediate density between both the vacuole and the Golgi complex. Pulse-chase labeling and fractionation of membranes indicated that p2CPY in this gradient region had already passed through the Golgi complex, which kinetically ordered it between the Golgi and the vacuole. A mutant CPY protein that lacks a functional vacuolar sorting signal was detected in Golgi fractions but not in the intermediate compartment indicating that this corresponds to a post-sorting compartment. Based on the low transport efficiency of the mutant CPY protein in vitro (decreased by sevenfold), this intermediate organelle most likely represents the donor compartment in our reconstitution assay. This organelle is not likely to be a transport vesicle intermediate because EM analysis indicates enrichment of 250-400 nm compartments and internalization of surface-bound 35S-alpha-factor at 15 degrees C resulted in its apparent cofractionation with wild-type p2CPY, indicating an endosome-like compartment (Singer, B., and H. Reizman. 1990. J. Cell Biol. 110:1911-1922). Fractionation of p2CPY accumulated in the temperature sensitive vps15 mutant revealed that the vps15 transport block did not occur in the endosome-like compartment but rather in the late Golgi complex, presumably the site of CPY sorting. Therefore, as seen in mammalian cells, yeast CPY is sorted away from secretory proteins in the late Golgi and transits to the vacuole via a distinct endosome-like intermediate.  相似文献   

7.
Membrane carboxypeptidase E (CPE) is a sorting receptor for targeting prohormones, such as pro-opiomelanocortin, to the regulated secretory pathway in endocrine cells. Its membrane association is necessary for it to bind a prohormone sorting signal at the trans-Golgi network (TGN) to facilitate targeting. In this study, we examined the lipid interaction of CPE in bovine pituitary secretory granule membranes, which are derived from the TGN. We show that CPE is associated with detergent-resistant lipid domains, or rafts, within secretory granule membranes. Lipid analysis revealed that these rafts are enriched in glycosphingolipids and cholesterol. Pulse-chase and subcellular fractionation experiments in AtT-20 cells show that the association of CPE with membrane rafts occurred only after it reached the Golgi. Cholesterol depletion resulted in dissociation of CPE from secretory granule membranes and decreased the binding of prohormones to membranes. In vivo cholesterol depletion using lovastatin resulted in the lack of sorting of CPE and its cargo to the regulated secretory pathway. We propose that the sorting receptor function of CPE necessitates its interaction with glycosphingolipid-cholesterol rafts at the TGN, thereby anchoring it in position to bind to its prohormone cargo.  相似文献   

8.
We have isolated four yeast mutants that are unable to partition maternal vacuoles into growing buds. Three of these vacuole segregation (vac) mutants also mislocalize the vacuolar protease carboxypeptidase Y (CPY) to the cell surface, a phenotype previously reported for vac strains. A fourth mutant, vac2-1, exhibits a temperature-sensitive defect in vacuole segregation but does not show a defect in protein targeting from the Golgi apparatus to the vacuole. Haploid vac2-1 cells grown at the non-permissive temperature do not secrete CPY or a second vacuolar protease, proteinase A (PrA). Furthermore, newly synthesized precursors of CPY are converted to mature forms with similar kinetics in both vac2-1 and wild-type cells. In addition, invertase is secreted normally from vac2-1 cells, indicating that post-Golgi steps in the secretory pathway are not blocked in this mutant. These results suggest that VAC2 function is necessary for vacuole division and segregation in yeast but is not involved in vacuole protein sorting events at the Golgi apparatus.  相似文献   

9.
The trans‐Golgi network (TGN) is a major sorting, packing and delivering station of newly synthesised proteins and lipids to their final destination. These cargo molecules follow the secretory pathway, which is a vital part of cellular trafficking machinery in all eukaryotic cells. This secretory pathway is well conserved in all eukaryotes from low‐level eukaryotes, such as yeast, to higher level eukaryotes like mammals. The molecular mechanisms of protein sorting by adaptor proteins, membrane elongation and transport to the final destinations by motor proteins and the cytoskeleton, and membrane pinching‐off by scission proteins must be choreographically managed for efficient cargo delivery, and the understanding of these detailed processes is not yet completed. Functionally, defects in these mechanisms are associated with the pathology of prominent diseases such as acute myeloid leukaemia, Charcot–Marie–Tooth disease, I‐cell disease and Wiskott–Aldrich syndrome. The present review points out the recent advances in our knowledge of the molecular mechanisms involved in the transportation of the cargo from the TGN towards the endosome.  相似文献   

10.
The trans-Golgi network (TGN) is the major sorting station in the secretory pathway of all eukaryotic cells. How the TGN sorts proteins and lipids to generate the enrichment of sphingolipids and sterols at the plasma membrane is poorly understood. To address this fundamental question in membrane trafficking, we devised an immunoisolation procedure for specific recovery of post-Golgi secretory vesicles transporting a transmembrane raft protein from the TGN to the cell surface in the yeast Saccharomyces cerevisiae. Using a novel quantitative shotgun lipidomics approach, we could demonstrate that TGN sorting selectively enriched ergosterol and sphingolipid species in the immunoisolated secretory vesicles. This finding, for the first time, indicates that the TGN exhibits the capacity to sort membrane lipids. Furthermore, the observation that the immunoisolated vesicles exhibited a higher membrane order than the late Golgi membrane, as measured by C-Laurdan spectrophotometry, strongly suggests that lipid rafts play a role in the TGN-sorting machinery.  相似文献   

11.
We have isolated cis-acting mutations in the gene encoding the yeast vacuolar protein carboxypeptidase Y (CPY) that result in missorting and aberrant secretion of up to 95% of newly synthesized CPY. The CPY polypeptides synthesized by these mutants use the late secretory pathway to exit the cell, since the late-acting sec1 mutation prevents their secretion. The mutant versions of CPY are secreted as the proCPY zymogen and are enzymatically activatable in vivo and in vitro. All the mutations, including small deletions and an amino acid substitution, map to the amino-terminal propeptide region and define a discrete yeast vacuolar localization domain whose integrity is required for efficient sorting of the CPY zymogen. Thus, the N-terminal propeptide of CPY carries out at least three functions: it mediates translocation across the endoplasmic reticulum, renders the enzyme inactive during transit, and targets the molecule to the vacuole.  相似文献   

12.
Clathrin-coated vesicles mediate the transport of the soluble vacuolar protein CPY from the TGN to the endosomal/prevacuolar compartment. Surprisingly, CPY sorting is not affected in clathrin deletion mutant cells. Here, we have investigated the clathrin-independent pathway that allows CPY transport to the vacuole. We find that CPY transport is mediated by the endosome and requires normal trafficking of its sorting receptor, Vps10p, the steady state distribution of which is not altered in chc1 cells. In contrast, Vps10p accumulates at the cell surface in a chc1/end3 double mutant, suggesting that Vps10p is rerouted to the cell surface in the absence of clathrin. We used a chimeric protein containing the first 50 amino acids of CPY fused to a green fluorescent protein (CPY-GFP) to mimic CPY transport in chc1. In the absence of clathrin, CPY-GFP resides in the lumen of the vacuole as in wild-type cells. However, in chc1/sec6 double mutants, CPY-GFP is present in internal structures, possibly endosomal membranes, that do not colocalize with the vacuole. We propose that Vps10p must be transported to and retrieved from the plasma membrane to mediate CPY sorting to the vacuole in the absence of clathrin-coated vesicles. In this circumstance, precursor CPY may be captured by retrieved Vps10p in an early or late endosome, rather than as it normally is in the trans-Golgi, and delivered to the vacuole by the normal VPS gene-dependent process. Once relieved of cargo protein, Vps10p would be recycled to the trans-Golgi and then to the cell surface for further rounds of sorting.  相似文献   

13.
The branching of exocytic transport routes in both yeast and mammalian cells has complicated studies of the late secretory pathway, and the mechanisms involved in exocytic cargo sorting and exit from the Golgi and endosomes are not well understood. Because cargo can be sorted away from a blocked route and secreted by an alternate route, mutants defective in only one route do not exhibit a strong secretory phenotype and are therefore difficult to isolate. In a genetic screen designed to isolate such mutants, we identified a novel conserved protein, Avl9p, the absence of which conferred lethality in a vps1Delta apl2Delta strain background (lacking a dynamin and an adaptor-protein complex 1 subunit). Depletion of Avl9p in this strain resulted in secretory defects as well as accumulation of Golgi-like membranes. The triple mutant also had a depolarized actin cytoskeleton and defects in polarized secretion. Overexpression of Avl9p in wild-type cells resulted in vesicle accumulation and a post-Golgi defect in secretion. Phylogenetic analysis indicated evolutionary relationships between Avl9p and regulators of membrane traffic and actin function.  相似文献   

14.
T R Graham  P A Scott    S D Emr 《The EMBO journal》1993,12(3):869-877
We have found that brefeldin A (BFA) inhibited the growth of an ise1 mutant of Saccharomyces cerevisiae. Genetic complementation and mapping studies demonstrated that ise1 was allelic to erg6, a gene required for the biosynthesis of the principal membrane sterol of yeast, ergosterol. Treatment of ise1 cells with BFA resulted in an immediate block in protein transport through the secretory pathway. Vacuolar carboxypeptidase Y (CPY) and the secreted pheromone alpha-factor accumulated as both the core glycosylated (ER) and alpha 1,6 mannosylated (early Golgi) forms in drug-treated cells. The modification of alpha-factor with alpha 1,6 mannose in BFA-treated cells did not appear to result from retrograde transport of the alpha 1,6 mannosyl-transferase into the ER. We found that transport of CPY from medial and late Golgi compartments to the vacuole was unaffected by BFA, nor was secretion of alpha 1,3 mannosylated alpha-factor or invertase blocked by BFA. The effects of BFA on the secretory pathway were also reversible after brief exposure (< 40 min) to the drug. We suggest that the primary effect of BFA in S. cerevisiae is restricted to the ER and the alpha 1,6 mannosyltransferase compartment of the Golgi complex.  相似文献   

15.
Whyte JR  Munro S 《Current biology : CB》2001,11(13):1074-1078
The soluble hydrolases of the mammalian lysosome are marked for delivery to this organelle by the addition of mannose 6-phosphate to their N-glycans. Two related mannose 6-phosphate receptors (MPRs) recognize this feature in the trans Golgi network (TGN) and deliver the hydrolases to the late endosome. In contrast, the vacuolar hydrolases of the yeast Saccharomyces cerevisiae do not contain 6-phosphate monoesters on their N-glycans, and the only sorting receptor so far identified in this organism is the product of the VPS10 gene. This protein also cycles between the Golgi and the late endosome, but is unrelated to the vertebrate MPRs, and recognizes a specific amino acid sequence of carboxypeptidase Y (CPY). This has led to the notion that although yeast and mammals share many components in Golgi to endosome traffic, they use unrelated receptor systems to sort their abundant soluble hydrolases. In this paper, we report that the yeast genome does in fact contain an uncharacterized ORF (YPR079w) that encodes a membrane protein that is distantly related to mammalian MPRs. The protein encoded by this gene (which we term MRL1) cycles through the late endosome. Moreover, there is a strong synergistic effect on the maturation of proteinases A and B when both MRL1 and VPS10 are deleted, which suggests that Mrl1p may serve as a sorting receptor in the delivery of vacuolar hydrolases.  相似文献   

16.
Transport between the trans-Golgi network (TGN) and late endosome represents a conserved, clathrin-dependent sorting event that separates lysosomal from secretory cargo molecules and is also required for localization of integral membrane proteins to the TGN. Previously, we reported a cell-free reaction that reconstitutes transport from the yeast TGN to the late endosome/prevacuolar compartment (PVC) and requires the PVC t-SNARE Pep12p. Here, we report that factors required both for formation of clathrin-coated vesicles at the TGN (the Chc1p clathrin heavy chain and the Vps1p dynamin homolog) and for vesicle fusion at the PVC (the Vps21p rab protein and Vps45p SM (Sec1/Munc18) protein) are required for cell-free transport. The marker for TGN-PVC transport, Kex2p, is initially present in a clathrin-containing membrane compartment that is competent for delivery of Kex2p to the PVC. A Kex2p chimera containing the cytosolic tail (C-tail) of the vacuolar protein sorting receptor, Vps10p, is also efficiently transported to the PVC. Antibodies against the Kex2p and Vps10p C-tails selectively block transport of Kex2p and the Kex2-Vps10p chimera. The requirements for factors involved in vesicle formation and fusion, the identification of the donor compartment as a clathrin-containing membrane, and the need for accessibility of C-tail sequences argue that the TGN-PVC transport reaction involves selective incorporation of TGN cargo molecules into clathrin-coated vesicle intermediates. Further biochemical dissection of this reaction should help elucidate the molecular requirements and hierarchy of events in TGN-to-PVC sorting and transport.  相似文献   

17.
The disulfide-bonded loop of chromogranin B (CgB), a regulated secretory protein with widespread distribution in neuroendocrine cells, is known to be essential for the sorting of CgB from the trans-Golgi network (TGN) to immature secretory granules. Here we show that this loop, when fused to the constitutively secreted protein alpha1-antitrypsin (AT), is sufficient to direct the fusion protein to secretory granules. Importantly, the sorting efficiency of the AT reporter protein bearing two loops (E2/3-AT-E2/3) is much higher compared with that of AT with a single disulfide-bonded loop. In contrast to endogenous CgB, E2/3-AT-E2/3 does not undergo Ca2+/pH-dependent aggregation in the TGN. Furthermore, the disulfide-bonded loop of CgB mediates membrane binding in the TGN and does so with 5-fold higher efficiency if two loops are present on the reporter protein. The latter finding supports the concept that under physiological conditions, aggregates of CgB are the sorted units of cargo which have multiple loops on their surface leading to high membrane binding and sorting efficiency of CgB in the TGN.  相似文献   

18.
Components involved in vesicle trafficking processes such as secretion, endocytosis, and autophagy are gaining recognition as important regulators and effectors of target of rapamycin (TOR) signaling. A recent report by now implicates Pmr1, a secretory pathway Ca(2+)/Mn(2+) ATPase located in the Golgi apparatus, as a novel regulator of TOR and its downstream targets in yeast.  相似文献   

19.
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.  相似文献   

20.
The trans-Golgi Network (TGN) sorts molecular “addresses” and sends newly synthesized proteins to their destination via vesicular transport carriers. Despite the functional significance of packaging processes at the TGN, the sorting of soluble proteins remains poorly understood. Recent research has shown that the Golgi resident protein Cab45 is a significant regulator of secretory cargo sorting at the TGN. Cab45 oligomerizes upon transient Ca2+ influx, recruits soluble cargo molecules (clients), and packs them in sphingomyelin-rich transport carriers. However, the identity of client molecules packed into Cab45 vesicles is scarce. Therefore, we used a precise and highly efficient secretome analysis technology called hiSPECs. Intriguingly, we observed that Cab45 deficient cells manifest hypersecretion of lysosomal hydrolases. Specifically, Cab45 deficient cells secrete the unprocessed precursors of prosaposin (PSAP) and progranulin (PGRN). In addition, lysosomes in these cells show an aberrant perinuclear accumulation suggesting a new role of Cab45 in lysosomal positioning. This work uncovers a yet unknown function of Cab45 in regulating lysosomal function.  相似文献   

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