Early/recycling endosomes-to-TGN transport involves two SNARE complexes and a Rab6 isoform.
(1/23)
The molecular mechanisms underlying early/recycling endosomes-to-TGN transport are still not understood. We identified interactions between the TGN-localized putative t-SNAREs syntaxin 6, syntaxin 16, and Vti1a, and two early/recycling endosomal v-SNAREs, VAMP3/cellubrevin, and VAMP4. Using a novel permeabilized cell system, these proteins were functionally implicated in the post-Golgi retrograde transport step. The function of Rab6a' was also required, whereas its closely related isoform, Rab6a, has previously been implicated in Golgi-to-endoplasmic reticulum transport. Thus, our study shows that membrane exchange between the early endocytic and the biosynthetic/secretory pathways involves specific components of the Rab and SNARE machinery, and suggests that retrograde transport between early/recycling endosomes and the endoplasmic reticulum is critically dependent on the sequential action of two members of the Rab6 subfamily. (+info)
How Tlg2p/syntaxin 16 'snares' Vps45.
(2/23)
Soluble N-ethylmaleimide sensitive factor-attachment protein receptors (SNAREs) and Sec1p/Munc18-homologs (SM proteins) play key roles in intracellular membrane fusion. The SNAREs form tight four-helix bundles (core complexes) that bring the membranes together, but it is unclear how this activity is coupled to SM protein function. Studies of the yeast trans-Golgi network (TGN)/endosomal SNARE complex, which includes the syntaxin-like SNARE Tlg2p, have suggested that its assembly requires activation by binding of the SM protein Vps45p to the cytoplasmic region of Tlg2p folded into a closed conformation. Nuclear magnetic resonance and biochemical experiments now show that Tlg2p and Pep12p, a late- endosomal syntaxin that interacts functionally but not directly with Vps45p, have a domain structure characteristic of syntaxins but do not adopt a closed conformation. Tlg2p binds tightly to Vps45p via a short N-terminal peptide motif that is absent in Pep12p. The Tlg2p/Vps45p binding mode is shared by the mammalian syntaxin 16, confirming that it is a Tlg2p homolog, and resembles the mode of interaction between the SM protein Sly1p and the syntaxins Ufe1p and Sed5p. Thus, this mechanism represents the most widespread mode of coupling between syntaxins and SM proteins. (+info)
Drosophila syntaxin 16 is a Q-SNARE implicated in Golgi dynamics.
(3/23)
SNARE isoforms appear to regulate specific intracellular membrane trafficking steps. To identify new SNARE proteins in Drosophila melanogaster we used a yeast two-hybrid screen to search for proteins that interact with SNAP. Here we report the identification of the Drosophila homologue of syntaxin 16. dsyntaxin 16 binds SNAP in a concentration-dependent fashion and genetically interacts with NSF2. Like its mammalian homologue, dsyntaxin 16 is ubiquitously expressed and appears to be localized to the Golgi apparatus. In addition, membranes containing dsyntaxin 16 become aggregated upon Brefeldin A treatment and are dispersed during meiosis. Inhibition of dsyntaxin 16 function by overexpression of truncated forms in cultured Schneider cells indicates that dsyntaxin 16 may selectively regulate Golgi dynamics. (+info)
GLUT4 recycles via a trans-Golgi network (TGN) subdomain enriched in Syntaxins 6 and 16 but not TGN38: involvement of an acidic targeting motif.
(4/23)
Insulin stimulates glucose transport in fat and muscle cells by triggering exocytosis of the glucose transporter GLUT4. To define the intracellular trafficking of GLUT4, we have studied the internalization of an epitope-tagged version of GLUT4 from the cell surface. GLUT4 rapidly traversed the endosomal system en route to a perinuclear location. This perinuclear GLUT4 compartment did not colocalize with endosomal markers (endosomal antigen 1 protein, transferrin) or TGN38, but showed significant overlap with the TGN target (t)-soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) Syntaxins 6 and 16. These results were confirmed by vesicle immunoisolation. Consistent with a role for Syntaxins 6 and 16 in GLUT4 trafficking we found that their expression was up-regulated significantly during adipocyte differentiation and insulin stimulated their movement to the cell surface. GLUT4 trafficking between endosomes and trans-Golgi network was regulated via an acidic targeting motif in the carboxy terminus of GLUT4, because a mutant lacking this motif was retained in endosomes. We conclude that GLUT4 is rapidly transported from the cell surface to a subdomain of the trans-Golgi network that is enriched in the t-SNAREs Syntaxins 6 and 16 and that an acidic targeting motif in the C-terminal tail of GLUT4 plays an important role in this process. (+info)
A novel STX16 deletion in autosomal dominant pseudohypoparathyroidism type Ib redefines the boundaries of a cis-acting imprinting control element of GNAS.
(5/23)
A unique heterozygous 3-kb microdeletion within STX16, a closely linked gene centromeric of GNAS, was previously identified in multiple unrelated kindreds as a cause of autosomal dominant pseudohypoparathyroidism type Ib (AD-PHP-Ib). We now report a novel heterozygous 4.4-kb microdeletion in a large kindred with AD-PHP-Ib. Affected individuals from this kindred share an epigenetic defect that is indistinguishable from that observed in patients with AD-PHP-Ib who carry the 3-kb microdeletion in the STX16 region (i.e., an isolated loss of methylation at GNAS exon A/B). The novel 4.4-kb microdeletion overlaps with the previously identified deletion by 1,286 bp and, similar to the latter deletion, removes several exons of STX16 (encoding syntaxin-16). Because these microdeletions lead to AD-PHP-Ib only after maternal transmission, we analyzed expression of this gene in lymphoblastoid cells of affected individuals with the 3-kb or the 4.4-kb microdeletion, an individual with a NESP55 deletion, and a healthy control. We found that STX16 mRNA was expressed in all cases from both parental alleles. Thus, STX16 is apparently not imprinted, and a loss-of-function mutation in one allele is therefore unlikely to be responsible for this disorder. Instead, the region of overlap between the two microdeletions likely harbors a cis-acting imprinting control element that is necessary for establishing and/or maintaining methylation at GNAS exon A/B, thus allowing normal G alpha(s) expression in the proximal renal tubules. In the presence of either of the two microdeletions, parathyroid hormone resistance appears to develop over time, as documented in an affected individual who was diagnosed at birth with the 4.4-kb deletion of STX16 and who had normal serum parathyroid hormone levels until the age of 21 mo. (+info)
Syntaxin 16 and syntaxin 5 are required for efficient retrograde transport of several exogenous and endogenous cargo proteins.
(6/23)
Retrograde transport allows proteins and lipids to leave the endocytic pathway to reach other intracellular compartments, such as trans-Golgi network (TGN)/Golgi membranes, the endoplasmic reticulum and, in some instances, the cytosol. Here, we have used RNA interference against the SNARE proteins syntaxin 5 and syntaxin 16, combined with recently developed quantitative trafficking assays, morphological approaches and cell intoxication analysis to show that these SNARE proteins are not only required for efficient retrograde transport of Shiga toxin, but also for that of an endogenous cargo protein - the mannose 6-phosphate receptor - and for the productive trafficking into cells of cholera toxin and ricin. We have found that the function of syntaxin 16 was specifically required for, and restricted to, the retrograde pathway. Strikingly, syntaxin 5 RNA interference protected cells particularly strongly against Shiga toxin. Since our trafficking analysis showed that apart from inhibiting retrograde endosome-to-TGN transport, the silencing of syntaxin 5 had no additional effect on Shiga toxin endocytosis or trafficking from TGN/Golgi membranes to the endoplasmic reticulum, we hypothesize that syntaxin 5 also has trafficking-independent functions. In summary, our data demonstrate that several cellular and exogenous cargo proteins use elements of the same SNARE machinery for efficient retrograde transport between early/recycling endosomes and TGN/Golgi membranes. (+info)
A syntaxin 10-SNARE complex distinguishes two distinct transport routes from endosomes to the trans-Golgi in human cells.
(7/23)