Membrane fusion in cells involves the interaction of SNARE proteins on apposing membranes. Formation of SNARE complexes is preceded by tethering events, and a number of protein complexes that are thought to mediate this have been identified. The VFT or GARP complex is required for endosome-Golgi traffic in yeast. It consists of four subunits, one of which, Vps51, has been shown to bind specifically to the SNARE Tlg1, which participates in the same fusion event. We have determined the structure of the N-terminal domain of Tlg1 bound to a peptide from the N terminus of Vps51. Binding depends mainly on residues 18-30 of Vps51. These form a short helix which lies in a conserved groove in the three-helix bundle formed by Tlg1. Surprisingly, although both Vps51 and Tlg1 are required for transport to the late Golgi from endosomes, removal of the Tlg1-binding sequences from Vps51 does not block such traffic in vivo. Thus, this particular interaction cannot be crucial to the process of vesicle docking or fusion. 相似文献
The Golgi associated retrograde protein complex (GARP) or Vps fifty-three (VFT) complex is part of cellular inter-compartmental transport systems. Here we report the identification of the VFT tethering factor complex and its interactions in mammalian cells. Subcellular fractionation shows that human Vps proteins are found in the smooth membrane/Golgi fraction but not in the cytosol. Immunostaining of human Vps proteins displays a vesicular distribution most concentrated at the perinuclear envelope. Co-staining experiments with endosomal markers imply an endosomal origin of these vesicles. Significant accumulation of VFT complex positive endosomes is found in the vicinity of the Trans Golgi Network area. This is in accordance with a putative role in Golgi associated transport processes. In Saccharomyces cerevisiae, GARP is the main effector of the small GTPase Ypt6p and interacts with the SNARE Tlg1p to facilitate membrane fusion. Accordingly, the human homologue of Ypt6p, Rab6, specifically binds hVps52. In human cells, the "orphan" SNARE Syntaxin 10 is the genuine binding partner of GARP mediated by hVps52. This reveals a previously unknown function of human Syntaxin 10 in membrane docking and fusion events at the Golgi. Taken together, GARP shows significant conservation between various species but diversification and specialization result in important differences in human cells. 相似文献
The biosynthetic pathway carries cargos from the endoplasmic reticulum (ER) to the trans Golgi network (TGN) via a typical passage through the Golgi. Interestingly, large particles such as procollagen, chylomicrons and some viruses all reach the TGN by atypical routes. Given this dichotomy, we anticipated that such cargos might rely on non-classical machineries downstream of the TGN. Using Herpes simplex virus type 1 (HSV-1) as a model and a synchronized infection protocol that focuses on TGN to plasma membrane transport, the present study revealed the surprising implication of the cellular serine-threonine protein kinase D in HSV-1 egress. These findings, confirmed by a variety of complementary means [pharmacological inhibitors, dominant negative mutant, RNA interference and electron microscopy (EM)], identify one of possibly several cellular factors that modulate the egress of viruses transiting at the TGN. Moreover, the involvement of this kinase, previously known to regulate the transport of small basolateral cargos, highlights the trafficking of both small and exceptionally large entities by a common machinery downstream of the TGN, in sharp contrast to earlier steps of transport. Conceptually, this indicates the TGN is not only a sorting station from which cargos can depart towards different destinations but also a meeting point where conventional and unconventional routes can meet along the biosynthetic pathway. Lastly, given the apical release of HSV-1 in neurons, it opens up the possibility that this kinase might regulate some apical sorting. 相似文献
ARF‐GTPases are important proteins that control membrane trafficking events. Their activity is largely influenced by the interplay between guanine nucleotide exchange factors (GEFs) and GTPase‐activating proteins (GAPs), which facilitate the activation or inactivation of ARF‐GTPases, respectively. There are 15 predicted proteins that contain an ARF‐GAP domain within the Arabidopsis thaliana genome, and these are classified as ARF‐GAP domain (AGD) proteins. The function and subcellular distribution of AGDs, including the ability to activate ARF‐GTPases in vivo, that remain largely uncharacterized to date. Here we show that AGD5 is localised to the trans‐Golgi network (TGN), where it co‐localises with ARF1, a crucial GTPase that is involved in membrane trafficking and which was previously shown to be distributed on Golgi and post‐Golgi structures of unknown nature. Taking advantage of the in vivo AGD5–ARF1 interaction at the TGN, we show that mutation of an arginine residue that is critical for ARF‐GAP activity of AGD5 leads to longer residence of ARF1 on the membranes, as expected if GTP hydrolysis on ARF1 was impaired due to a defective GAP. Our results establish the nature of the post‐Golgi compartments in which ARF1 localises, as well as identifying the role of AGD5 in vivo as a TGN‐localised GAP. Furthermore, in vitro experiments established the promiscuous interaction between AGD5 and the plasma membrane‐localised ADP ribosylation factor B (ARFB), confirming that ARF‐GAP specificity for ARF‐GTPases within the cell environment may be spatially regulated. 相似文献
The trans‐Golgi network (TGN) is a major site for sorting of cargo to either the vacuole or apoplast. The TGN‐localized coiled‐coil protein TNO1 is a putative tethering factor that interacts with the TGN t‐SNARE SYP41 and is required for correct localization of the SYP61 t‐SNARE. An Arabidopsis thaliana tno1 mutant is hypersensitive to salt stress and partially mislocalizes vacuolar proteins to the apoplast, indicating a role in vacuolar trafficking. Here, we show that overexpression of SYP41 or SYP61 significantly increases SYP41–SYP61 complex formation in a tno1 mutant, and rescues the salt sensitivity and defective vacuolar trafficking of the tno1 mutant. The TGN is disrupted and vesicle budding from Golgi cisternae is reduced in the tno1 mutant, and these defects are also rescued by overexpression of SYP41 or SYP61. Our results suggest that the trafficking and Golgi morphology defects caused by loss of TNO1 can be rescued by increasing SYP41–SYP61 t‐SNARE complex formation, implicating TNO1 as a tethering factor mediating efficient vesicle fusion at the TGN. 相似文献
Arabidopsis thaliana PHO1 is primarily expressed in the root vascular cylinder and is involved in the transfer of inorganic phosphate (Pi) from roots to shoots. To analyze the role of PHO1 in transport of Pi, we have generated transgenic plants expressing PHO1 in ectopic A. thaliana tissues using an estradiol-inducible promoter. Leaves treated with estradiol showed strong PHO1 expression, leading to detectable accumulation of PHO1 protein. Estradiol-mediated induction of PHO1 in leaves from soil-grown plants, in leaves and roots of plants grown in liquid culture, or in leaf mesophyll protoplasts, was all accompanied by the specific release of Pi to the extracellular medium as early as 2-3 h after addition of estradiol. Net Pi export triggered by PHO1 induction was enhanced by high extracellular Pi and weakly inhibited by the proton-ionophore carbonyl cyanide m-chlorophenylhydrazone. Expression of a PHO1-GFP construct complementing the pho1 mutant revealed GFP expression in punctate structures in the pericycle cells but no fluorescence at the plasma membrane. When expressed in onion epidermal cells or in tobacco mesophyll cells, PHO1-GFP was associated with similar punctate structures that co-localized with the Golgi/trans-Golgi network and uncharacterized vesicles. However, PHO1-GFP could be partially relocated to the plasma membrane in leaves infiltrated with a high-phosphate solution. Together, these results show that PHO1 can trigger Pi export in ectopic plant cells, strongly indicating that PHO1 is itself a Pi exporter. Interestingly, PHO1-mediated Pi export was associated with its localization to the Golgi and trans-Golgi networks, revealing a role for these organelles in Pi transport. 相似文献
Retrograde transport between endosomes and the trans-Golgi network (TGN) is essential for the recycling of membrane proteins which are involved in a range of biological processes. A variety of machinery components have been identified at the TGN which regulate endosome-to-TGN transport, including small G proteins, SNAREs, tethering factors and scaffold molecules. The challenge is to understand how these regulatory components orchestrate not only the specific docking and fusion of retrograde membrane carriers with the TGN, but also maintain the integrity of this highly dynamic compartment to ensure efficient delivery and export of cargo. Here we review recent advances in defining the form and function of tethers and scaffolds in the regulation of the retrograde transport pathways. 相似文献
How polytopic plasma membrane (PM) proteins reach their destination in plant cells remains elusive. Using transgenic tobacco BY-2 cells, we previously showed that the rice secretory carrier membrane protein 1 (SCAMP1), an integral membrane protein with four transmembrane domains (TMDs), is localized to the PM and trans-Golgi network (TGN). Here, we study the transport pathway and sorting signals of SCAMP1 by following its transient expression in tobacco BY-2 protoplasts and show that SCAMP1 reaches the PM via an endoplasmic reticulum (ER)-Golgi-TGN-PM pathway. Loss-of-function and gain-of-function analysis of various green fluorescent protein (GFP) fusions with SCAMP1 mutations further demonstrates that: (i) the cytosolic N-terminus of SCAMP1 contains an ER export signal; (ii) the transmembrane domain 2 (TMD2) and TMD3 of SCAMP1 are essential for Golgi export; (iii) SCAMP1 TMD1 is essential for TGN-to-PM targeting; (iv) the predicted topology of SCAMP1 and its various mutants remain identical as demonstrated by protease protection assay. Therefore, both the cytosolic N-terminus and TMD sequences of SCAMP1 play integral roles in mediating its transport to the PM via an ER-Golgi-TGN pathway. 相似文献
Cargo adaptors control intracellular trafficking of transmembrane proteins by sorting them into membrane transport carriers. The COPI, COPII, and clathrin cargo adaptors are structurally well characterized, but other cargo adaptors remain poorly understood. Exomer is a specialized cargo adaptor that sorts specific proteins into trans‐Golgi network (TGN)‐derived vesicles in response to cellular signals. Exomer is recruited to the TGN by the Arf1 GTPase, a universally conserved trafficking regulator. Here, we report the crystal structure of a tetrameric exomer complex composed of two copies each of the Chs5 and Chs6 subunits. The structure reveals the FN3 and BRCT domains of Chs5, which together we refer to as the FBE domain (F N3–B RCT of e xomer), project from the exomer core complex. The overall architecture of the FBE domain is reminiscent of the appendage domains of other cargo adaptors, although it exhibits a distinct topology. In contrast to appendage domains, which bind accessory factors, we show that the primary role of the FBE domain is to bind Arf1 for recruitment of exomer to membranes. 相似文献
Enveloped viruses employ diverse and complex strategies for wrapping at cellular membranes, many of which are poorly understood. Here, an ultrastructural study of herpes simplex virus 1 (HSV1)‐infected cells revealed envelopment in tubular membranes. These tubules were labelled by the fluid phase marker horseradish peroxidase (HRP), and were observed to wrap capsids as early as 2 min after HRP addition, indicating that the envelope had recently cycled from the cell surface. Consistent with this, capsids did not colocalise with either the trans‐Golgi network marker TGN46 or late endosomal markers, but showed coincidence with the transferrin receptor. Virus glycoproteins were retrieved from the plasma membrane (PM) to label wrapping capsids, a process that was dependent on both dynamin and Rab5. Combined depletion of Rab5 and Rab11 reduced virus yield to <1%, resulting in aberrant localisation of capsids. These results suggest that endocytosis from the PM into endocytic tubules provides the main source of membrane for HSV1, and reveal a new mechanism for virus exploitation of the endocytic pathway. 相似文献
The trimeric Vps29-Vps35-Vps26 sub-complex of retromer mediates retrograde transport of transmembrane proteins from endosomes to the trans-Golgi network. Our group has recently identified a Vps26 paralogue, Vps26B, which is able to suppress the expression of Vps26A when exogenously expressed in mammalian cells and defines a distinct retromer complex (Vps26B-retromer) in vivo and in vitro. In this study, we use HEK293 cells stably expressing either Vps26A-myc or Vps26B-myc to address the role of retromer cargo transport and subcellular localization of the two core retromer complexes as defined by the two mammalian-specific Vps26 paralogues. Vps26B-retromer, like Vps26A-retromer, associates with TBC1D5 and GOLPH3. In contrast, no interaction between Vps26B-retromer and cation-independent mannose 6-phosphate receptor (CI-M6PR) was detected, leading to a degradation of this receptor and an increase in cathepsin D secretion. Colocalization of Vps26 paralogues with different endosomally located Rab proteins shows prolonged association of Vps26B-retromer with maturing endosomes relative to Vps26A-retromer. Interestingly, the cycling of CI-M6PR is restored upon deletion of the variable Vps26B C-terminal region indicating that this region is directly responsible for the differential function of the two paralogues. In summary, we show that the two distinct retromer complexes defined by different Vps26 paralogues are not functionally equivalent and that the Vps26B C-terminal region can control cargo selection of the Vps26B-retromer. 相似文献
Prasinophytes are a paraphyletic assemblage of nine heterogeneous lineages in the Chlorophyta clade of Archaeplastida. Until now, seven complete mitochondrial genomes have been sequenced from four prasinophyte lineages. Here, we report the mitochondrial genome of Pyramimonas parkeae, the first representative of the prasinophyte clade I. The circular‐mapping molecule is 43,294 bp long, AT rich (68.8%), very compact and it comprises two 6,671 bp long inverted repeat regions. The gene content is slightly smaller than the gene‐richest prasinophyte mitochondrial genomes. The single identified intron is located in the cytochrome c oxidase subunit 1 gene (cox1). Interestingly, two exons of cox1 are encoded on the same strand of DNA in the reverse order and the mature mRNA is formed by trans‐splicing. The phylogenetic analysis using the data set of 6,037 positions assembled from 34 mtDNA‐encoded proteins of 48 green algae and plants is not in compliance with the branching order of prasinophyte clades revealed on the basis of 18S rRNA genes and cpDNA‐encoded proteins. However, the phylogenetic analyses based on all three genomic elements support the sister position of prasinophyte clades Pyramimonadales and Mamiellales. 相似文献
The yeast SNX4 sub‐family of sorting nexin containing a Bin‐Amphiphysin‐Rvs domain (SNX‐BAR) proteins, Snx4/Atg24, Snx41 and Atg20/Snx42, are required for endocytic recycling and selective autophagy. Here, we show that Snx4 forms 2 functionally distinct heterodimers: Snx4‐Atg20 and Snx4‐Snx41. Each heterodimer coats an endosome‐derived tubule that mediates retrograde sorting of distinct cargo; the v‐SNARE, Snc1, is a cargo of the Snx4‐Atg20 pathway, and Snx4‐Snx41 mediates retrograde sorting of Atg27, an integral membrane protein implicated in selective autophagy. Live cell imaging of individual endosomes shows that Snx4 and the Vps5‐Vps17 retromer SNX‐BAR heterodimer operate concurrently on a maturing endosome. Consistent with this, the yeast dynamin family protein, Vps1, which was previously shown to promote fission of retromer‐coated tubules, promotes fission of Snx4‐Atg20 coated tubules. The results indicate that the yeast SNX‐BAR proteins coat 3 distinct types of endosome‐derived carriers that mediate endosome‐to‐Golgi retrograde trafficking. 相似文献
Processing of amyloid precursor protein (APP) into amyloid‐β peptide (Aβ) is crucial for the development of Alzheimer's disease (AD). Because this processing is highly dependent on its intracellular itinerary, altered subcellular targeting of APP is thought to directly affect the degree to which Aβ is generated. The sorting receptor SorCS1 has been genetically linked to AD, but the underlying molecular mechanisms are poorly understood. We analyze two SorCS1 variants; one, SorCS1c, conveys internalization of surface‐bound ligands whereas the other, SorCS1b, does not. In agreement with previous studies, we demonstrate co‐immunoprecipitation and co‐localization of both SorCS1 variants with APP. Our results suggest that SorCS1c and APP are internalized independently, although they mostly share a common post‐endocytic pathway. We introduce functional Venus‐tagged constructs to study SorCS1b and SorCS1c in living cells. Both variants are transported by fast anterograde axonal transport machinery and about 30% of anterograde APP‐positive transport vesicles contain SorCS1. Co‐expression of SorCS1b caused no change of APP transport kinetics, but SorCS1c reduced the anterograde transport rate of APP and increased the number of APP‐positive stationary vesicles. These data suggest that SorCS1 and APP share trafficking pathways and that SorCS1c can retain APP from insertion into anterograde transport vesicles.
Munc 18-1 is a member of the Sec/Munc family of syntaxin-binding proteins known to bind to the plasma membrane Q-SNARE syntaxin1 and whose precise role in regulated exocytosis remains controversial. Here, we show that Munc 18-1 plays a positive role in regulated insulin secretion from pancreatic beta cells. Munc 18-1 depletion caused a loss in the secretory capacity of both transiently transfected INS 1E cells and a stable clone with tetracycline-regulated Munc 18-1 RNA interference. In addition, Munc 18-1-depleted cells exhibited defective docking of insulin granules to the plasma membrane and accumulated insulin in the trans Golgi network. Furthermore, glucose stimulation after Munc 18-1 depletion resulted in the rapid formation of autophagosomes. In contrast, overexpression of Munc 18-1 had no effect on insulin secretion. Although there was no detectable interaction between Munc 18-1 and Munc-18-interacting protein 1 or calcium/calmodulin-dependent serine protein kinase, Munc 18-1 associated with the granular protein granuphilin. This association was regulated by glucose and was required for the specific interaction of insulin granules with syntaxin1. We conclude that Munc 18-1 and granuphilin collaborate in the docking of insulin granules to the plasma membrane in an initial fusion-incompetent state, with Munc 18-1 subsequently playing a positive role in a later stage of insulin granule exocytosis. 相似文献
Human Vps13 proteins are associated with several diseases, including the neurodegenerative disorder Chorea‐acanthocytosis (ChAc), yet the biology of these proteins is still poorly understood. Studies in Saccharomyces cerevisiae, Dictyostelium discoideum, Tetrahymena thermophila and Drosophila melanogaster point to the involvement of Vps13 in cytoskeleton organization, vesicular trafficking, autophagy, phagocytosis, endocytosis, proteostasis, sporulation and mitochondrial functioning. Recent findings show that yeast Vps13 binds to phosphatidylinositol lipids via 4 different regions and functions at membrane contact sites, enlarging the list of Vps13 functions. This review describes the great potential of simple eukaryotes to decipher disease mechanisms in higher organisms and highlights novel insights into the pathological role of Vps13 towards ChAc. 相似文献
Ferlins are a family of transmembrane‐anchored vesicle fusion proteins uniquely characterized by 5–7 tandem cytoplasmic C2 domains, Ca2+‐regulated phospholipid‐binding domains that regulate vesicle fusion in the synaptotagmin family. In humans, dysferlin mutations cause limb‐girdle muscular dystrophy type 2B (LGMD2B) due to defective Ca2+‐dependent, vesicle‐mediated membrane repair and otoferlin mutations cause non‐syndromic deafness due to defective Ca2+‐triggered auditory neurotransmission. In this study, we describe the tissue‐specific expression, subcellular localization and endocytic trafficking of the ferlin family. Studies of endosomal transit together with 3D‐structured illumination microscopy reveals dysferlin and myoferlin are abundantly expressed at the PM and cycle to Rab7‐positive late endosomes, supporting potential roles in the late‐endosomal pathway. In contrast, Fer1L6 shows concentrated localization to a specific compartment of the trans‐Golgi/recycling endosome, cycling rapidly between this compartment and the PM via Rab11 recycling endosomes. Otoferlin also shows trans‐Golgi to PM cycling, with very low levels of PM otoferlin suggesting either brief PM residence, or rare incorporation of otoferlin molecules into the PM. Thus, type‐I and type‐II ferlins segregate as PM/late‐endosomal or trans‐Golgi/recycling ferlins, consistent with different ferlins mediating vesicle fusion events in specific subcellular locations. 相似文献
