scholarly journals mTOR-mediated phosphorylation of VAMP8 and SCFD1 regulates autophagosome maturation

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Hong Huang ◽  
Qinqin Ouyang ◽  
Min Zhu ◽  
Haijia Yu ◽  
Kunrong Mei ◽  
...  
Keyword(s):  
Complex Formation ◽  
Lipid Droplet ◽  
Mouse Liver ◽  
Mammalian Target Of Rapamycin ◽  
Snare Complex ◽  
Target Of Rapamycin ◽  
Protein Receptor ◽  
Autophagosome Maturation ◽  
Snare Complex Formation

AbstractThe mammalian target of rapamycin (mTORC1) has been shown to regulate autophagy at different steps. However, how mTORC1 regulates the N-ethylmaleimide-sensitive protein receptor (SNARE) complex remains elusive. Here we show that mTORC1 inhibits formation of the SNARE complex (STX17-SNAP29-VAMP8) by phosphorylating VAMP8, thereby blocking autophagosome-lysosome fusion. A VAMP8 phosphorylation mimic mutant is unable to promote autophagosome-lysosome fusion in vitro. Furthermore, we identify SCFD1, a Sec1/Munc18-like protein, that localizes to the autolysosome and is required for SNARE complex formation and autophagosome-lysosome fusion. VAMP8 promotes SCFD1 recruitment to autolysosomes when dephosphorylated. Consistently, phosphorylated VAMP8 or SCFD1 depletion inhibits autophagosome-lysosome fusion, and expression of phosphomimic VAMP8 leads to increased lipid droplet accumulation when expressed in mouse liver. Thus, our study supports that mTORC1-mediated phosphorylation of VAMP8 blocks SCFD1 recruitment, thereby inhibiting STX17-SNAP29-VAMP8 complex formation and autophagosome-lysosome fusion.

scholarly journals Dual inhibition of SNARE complex formation by tomosyn ensures controlled neurotransmitter release

2008 ◽  
Vol 183 (2) ◽  
pp. 323-337 ◽  
Author(s):  
Toshiaki Sakisaka ◽  
Yasunori Yamamoto ◽  
Sumiko Mochida ◽  
Michiko Nakamura ◽  
Kouki Nishikawa ◽  
...  
Keyword(s):  
Complex Formation ◽  
Neurotransmitter Release ◽  
Snare Complex ◽  
Protein Receptor ◽  
Repeat Domain ◽  
Dual Inhibition ◽  
Presynaptic Nerve Terminals ◽  
Synaptic Vesicle Fusion ◽  
Deficient Mice ◽  
Snare Complex Formation

Neurotransmitter release from presynaptic nerve terminals is regulated by soluble NSF attachment protein receptor (SNARE) complex–mediated synaptic vesicle fusion. Tomosyn inhibits SNARE complex formation and neurotransmitter release by sequestering syntaxin-1 through its C-terminal vesicle-associated membrane protein (VAMP)–like domain (VLD). However, in tomosyn-deficient mice, the SNARE complex formation is unexpectedly decreased. In this study, we demonstrate that the N-terminal WD-40 repeat domain of tomosyn catalyzes the oligomerization of the SNARE complex. Microinjection of the tomosyn N-terminal WD-40 repeat domain into neurons prevented stimulated acetylcholine release. Thus, tomosyn inhibits neurotransmitter release by catalyzing oligomerization of the SNARE complex through the N-terminal WD-40 repeat domain in addition to the inhibitory activity of the C-terminal VLD.

scholarly journals The tethering complex HOPS catalyzes assembly of the soluble SNARE Vam7 into fusogenic trans-SNARE complexes

2013 ◽  
Vol 24 (23) ◽  
pp. 3746-3753 ◽  
Author(s):  
Michael Zick ◽  
William Wickner
Keyword(s):  
Fusion Reaction ◽  
Snare Complex ◽  
Soluble Form ◽  
Protein Receptor ◽  
In Trans ◽  
Low Levels ◽  
Vacuolar Membranes ◽  
Snare Complex Formation ◽  
Indirect Stimulation

The fusion of yeast vacuolar membranes depends on the disassembly of cis–soluble N-ethylmaleimide–sensitive factor attachment protein receptor (SNARE) complexes and the subsequent reassembly of new SNARE complexes in trans. The disassembly of cis-SNARE complexes by Sec17/Sec18p releases the soluble SNARE Vam7p from vacuolar membranes. Consequently, Vam7p needs to be recruited to the membrane at future sites of fusion to allow the formation of trans-SNARE complexes. The multisubunit tethering homotypic fusion and vacuole protein sorting (HOPS) complex, which is essential for the fusion of vacuolar membranes, was previously shown to have direct affinity for Vam7p. The functional significance of this interaction, however, has been unclear. Using a fully reconstituted in vitro fusion reaction, we now show that HOPS facilitates membrane fusion by recruiting Vam7p for fusion. In the presence of HOPS, unlike with other tethering agents, very low levels of added Vam7p suffice to induce vigorous fusion. This is a specific recruitment of Vam7p rather than an indirect stimulation of SNARE complex formation through tethering, as HOPS does not facilitate fusion with a low amount of a soluble form of another vacuolar SNARE, Vti1p. Our findings establish yet another function among the multiple tasks that HOPS performs to catalyze the fusion of yeast vacuoles.

scholarly journals rsly1 Binding to Syntaxin 5 Is Required for Endoplasmic Reticulum-to-Golgi Transport but Does Not Promote SNARE Motif Accessibility

2004 ◽  
Vol 15 (1) ◽  
pp. 162-175 ◽  
Author(s):  
Antionette L. Williams ◽  
Sebastian Ehm ◽  
Noëlle C. Jacobson ◽  
Dalu Xu ◽  
Jesse C. Hay
Keyword(s):  
Endoplasmic Reticulum ◽  
Complex Formation ◽  
Protein Function ◽  
Snare Complex ◽  
Golgi Transport ◽  
Snare Motif ◽  
Sm Protein ◽  
Snare Complex Formation

Although some of the principles of N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) function are well understood, remarkably little detail is known about sec1/munc18 (SM) protein function and its relationship to SNAREs. Popular models of SM protein function hold that these proteins promote or maintain an open and/or monomeric pool of syntaxin molecules available for SNARE complex formation. To address the functional relationship of the mammalian endoplasmic reticulum/Golgi SM protein rsly1 and its SNARE binding partner syntaxin 5, we produced a conformation-specific monoclonal antibody that binds only the available, but not the cis-SNARE–complexed nor intramolecularly closed form of syntaxin 5. Immunostaining experiments demonstrated that syntaxin 5 SNARE motif availability is nonuniformly distributed and focally regulated. In vitro endoplasmic reticulum-to-Golgi transport assays revealed that rsly1 was acutely required for transport, and that binding to syntaxin 5 was absolutely required for its function. Finally, manipulation of rsly1–syntaxin 5 interactions in vivo revealed that they had remarkably little impact on the pool of available syntaxin 5 SNARE motif. Our results argue that although rsly1 does not seem to regulate the availability of syntaxin 5, its function is intimately associated with syntaxin binding, perhaps promoting a later step in SNARE complex formation or function.

scholarly journals An intramolecular t-SNARE complex functions in vivo without the syntaxin NH2-terminal regulatory domain

2006 ◽  
Vol 172 (2) ◽  
pp. 295-307 ◽  
Author(s):  
Jeffrey S. Van Komen ◽  
Xiaoyang Bai ◽  
Brenton L. Scott ◽  
James A. McNew
Keyword(s):  
Plasma Membrane ◽  
Complex Formation ◽  
Secretory Pathway ◽  
Regulatory Element ◽  
Snare Complex ◽  
Regulatory Domain ◽  
Vesicle Membrane ◽  
Snare Complex Formation

Membrane fusion in the secretory pathway is mediated by SNAREs (located on the vesicle membrane [v-SNARE] and the target membrane [t-SNARE]). In all cases examined, t-SNARE function is provided as a three-helix bundle complex containing three ∼70–amino acid SNARE motifs. One SNARE motif is provided by a syntaxin family member (the t-SNARE heavy chain), and the other two helices are contributed by additional t-SNARE light chains. The syntaxin family is the most conformationally dynamic group of SNAREs and appears to be the major focus of SNARE regulation. An NH2-terminal region of plasma membrane syntaxins has been assigned as a negative regulatory element in vitro. This region is absolutely required for syntaxin function in vivo. We now show that the required function of the NH2-terminal regulatory domain (NRD) of the yeast plasma membrane syntaxin, Sso1p, can be circumvented when t-SNARE complex formation is made intramolecular. Our results suggest that the NRD is required for efficient t-SNARE complex formation and does not recruit necessary scaffolding factors.

scholarly journals Sly1 protein bound to Golgi syntaxin Sed5p allows assembly and contributes to specificity of SNARE fusion complexes

2002 ◽  
Vol 157 (4) ◽  
pp. 645-655 ◽  
Author(s):  
Renwang Peng ◽  
Dieter Gallwitz
Keyword(s):  
Complex Formation ◽  
Snare Complex ◽  
Marked Contrast ◽  
Transport Vesicles ◽  
Target Membrane ◽  
First Time ◽  
Kinetics Of ◽  
Specific Membrane ◽  
Snare Complex Formation

Fusion of transport vesicles with their target organelles involves specific membrane proteins, SNAREs, which form tight complexes bridging the membranes to be fused. Evidence from yeast and mammals indicates that Sec1 family proteins act as regulators of membrane fusion by binding to the target membrane SNAREs. In experiments with purified proteins, we now made the observation that the ER to Golgi core SNARE fusion complex could be assembled on syntaxin Sed5p tightly bound to the Sec1-related Sly1p. Sly1p also bound to preassembled SNARE complexes in vitro and was found to be part of a vesicular/target membrane SNARE complex immunoprecipitated from yeast cell lysates. This is in marked contrast to the exocytic SNARE assembly in neuronal cells where high affinity binding of N-Sec1/Munc-18 to syntaxin 1A precluded core SNARE fusion complex formation. We also found that the kinetics of SNARE complex formation in vitro with either Sly1p-bound or free Sed5p was not significantly different. Importantly, several presumably nonphysiological SNARE complexes easily generated with Sed5p did not form when the syntaxin was first bound to Sly1p. This indicates for the first time that a Sec1 family member contributes to the specificity of SNARE complex assembly.

scholarly journals HOPS Initiates Vacuole Docking by Tethering Membranes before trans-SNARE Complex Assembly

2010 ◽  
Vol 21 (13) ◽  
pp. 2297-2305 ◽  
Author(s):  
Christopher M. Hickey ◽  
William Wickner
Keyword(s):  
Complex Formation ◽  
Present Model ◽  
Snare Complex ◽  
Snare Proteins ◽  
Membrane Association ◽  
Attachment Protein ◽  
Protein Receptor ◽  
Sensitive Factor ◽  
Rab Effector ◽  
Snare Complex Formation

Vacuole homotypic fusion has been reconstituted with all purified components: vacuolar lipids, four soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins, Sec17p, Sec18p, the Rab Ypt7p, and the hexameric homotypic fusion and vacuole protein sorting complex (HOPS). HOPS is a Rab-effector with direct affinity for SNAREs (presumably via its Sec1-Munc18 homologous subunit Vps33p) and for certain vacuolar lipids. Each of these pure vacuolar proteins was required for optimal proteoliposome clustering, raising the question of which was most directly involved. We now present model subreactions of clustering and fusion that reveal that HOPS is the direct agent of tethering. The Rab and vacuole lipids contribute to tethering by supporting the membrane association of HOPS. HOPS indirectly facilitates trans-SNARE complex formation by tethering membranes, because the synthetic liposome tethering factor polyethylene glycol can also stimulate trans-SNARE complex formation and fusion. SNAREs further stabilize the associations of HOPS-tethered membranes. HOPS then protects newly formed trans-SNARE complexes from disassembly by Sec17p/Sec18p.

Targeting mammalian target of rapamycin: prospects for the treatment of inflammatory bowel diseases

2020 ◽  
Vol 27 ◽  
Author(s):  
Naser-Aldin Lashgari ◽  
Nazanin Momeni Roudsari ◽  
Saeideh Momtaz ◽  
Negar Ghanaatian ◽  
Parichehr Kohansal ◽  
...  
Keyword(s):  
Signaling Pathway ◽  
Mammalian Target Of Rapamycin ◽  
Mtor Pathway ◽  
Mtor Signaling ◽  
Target Of Rapamycin ◽  
In Vivo Studies ◽  
Cochrane Library ◽  
Mtor Signaling Pathway ◽  
Inflammatory Bowel

: Inflammatory bowel disease (IBD) is a general term for a group of chronic and progressive disorders. Several cellular and biomolecular pathways are implicated in the pathogenesis of IBD, yet the etiology is unclear. Activation of the mammalian target of rapamycin (mTOR) pathway in the intestinal epithelial cells was also shown to induce inflammation. This review focuses on the inhibition of the mTOR signaling pathway and its potential application in treating IBD. We also provide an overview on plant-derived compounds that are beneficial for the IBD management through modulation of the mTOR pathway. Data were extracted from clinical, in vitro and in vivo studies published in English between 1995 and May 2019, which were collected from PubMed, Google Scholar, Scopus and Cochrane library databases. Results of various studies implied that inhibition of the mTOR signaling pathway downregulates the inflammatory processes and cytokines involved in IBD. In this context, a number of natural products might reverse the pathological features of the disease. Furthermore, mTOR provides a novel drug target for IBD. Comprehensive clinical studies are required to confirm the efficacy of mTOR inhibitors in treating IBD.

scholarly journals Synaptotagmin Expands Membrane Fusion Pore by Facilitating SNARE-Complex Formation

2011 ◽  
Vol 100 (3) ◽  
pp. 185a
Author(s):  
Jiajie Diao ◽  
Janghyun Yoo ◽  
Han-Ki Lee ◽  
Yoosoo Yang ◽  
Dae-Hyuk Kweon ◽  
...  
Keyword(s):  
Complex Formation ◽  
Membrane Fusion ◽  
Snare Complex ◽  
Fusion Pore ◽  
Snare Complex Formation

scholarly journals Sec1p and Mso1p C-terminal tails cooperate with the SNAREs and Sec4p in polarized exocytosis

2011 ◽  
Vol 22 (2) ◽  
pp. 230-244 ◽  
Author(s):  
Marion Weber-Boyvat ◽  
Nina Aro ◽  
Konstantin G. Chernov ◽  
Tuula Nyman ◽  
Jussi Jäntti
Keyword(s):  
Close Association ◽  
Adaptor Protein ◽  
Binding Mode ◽  
Snare Complex ◽  
Bimolecular Fluorescence Complementation ◽  
Rab Gtpase ◽  
Temperature Sensitive ◽  
Nucleotide Exchange ◽  
Protein Receptor

The Sec1/Munc18 protein family members perform an essential, albeit poorly understood, function in association with soluble n-ethylmaleimide sensitive factor adaptor protein receptor (SNARE) complexes in membrane fusion. The Saccharomyces cerevisiae Sec1p has a C-terminal tail that is missing in its mammalian homologues. Here we show that deletion of the Sec1p tail (amino acids 658–724) renders cells temperature sensitive for growth, reduces sporulation efficiency, causes a secretion defect, and abolishes Sec1p-SNARE component coimmunoprecipitation. The results show that the Sec1p tail binds preferentially ternary Sso1p-Sec9p-Snc2p complexes and it enhances ternary SNARE complex formation in vitro. The bimolecular fluorescence complementation (BiFC) assay results suggest that, in the SNARE-deficient sso2–1 Δsso1 cells, Mso1p, a Sec1p binding protein, helps to target Sec1p(1–657) lacking the C-terminal tail to the sites of secretion. The results suggest that the Mso1p C terminus is important for Sec1p(1–657) targeting. We show that, in addition to Sec1p, Mso1p can bind the Rab-GTPase Sec4p in vitro. The BiFC results suggest that Mso1p acts in close association with Sec4p on intracellular membranes in the bud. This association depends on the Sec4p guanine nucleotide exchange factor Sec2p. Our results reveal a novel binding mode between the Sec1p C-terminal tail and the SNARE complex, and suggest a role for Mso1p as an effector of Sec4p.

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