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Deficiency in the conserved oligomeric Golgi (COG) complex that orchestrates SNARE‐mediated tethering/fusion of vesicles that recycle the Golgi's glycosylation machinery results in severe glycosylation defects. Although two major Golgi v‐SNAREs, GS28/GOSR1, and GS15/BET1L, are depleted in COG‐deficient cells, the complete knockout of GS28 and GS15 only modestly affects Golgi glycosylation, indicating the existence of an adaptation mechanism in Golgi SNARE. Indeed, quantitative mass‐spectrometry analysis of STX5‐interacting proteins revealed two novel Golgi SNARE complexes—STX5/SNAP29/VAMP7 and STX5/VTI1B/STX8/YKT6. These complexes are present in wild‐type cells, but their usage is significantly increased in both GS28‐ and COG‐deficient cells. Upon GS28 deletion, SNAP29 increased its Golgi residency in a STX5‐dependent manner. While STX5 depletion and Retro2‐induced diversion from the Golgi severely affect protein glycosylation, GS28/SNAP29 and GS28/VTI1B double knockouts alter glycosylation similarly to GS28 KO, indicating that a single STX5‐based SNARE complex is sufficient to support Golgi glycosylation. Importantly, co‐depletion of three Golgi SNARE complexes in GS28/SNAP29/VTI1B TKO cells resulted in severe glycosylation defects and a reduced capacity for glycosylation enzyme retention at the Golgi. This study demonstrates the remarkable plasticity in SXT5‐mediated membrane trafficking, uncovering a novel adaptive response to the failure of canonical intra‐Golgi vesicle tethering/fusion machinery. [ABSTRACT FROM AUTHOR]

Intracellular membrane protein trafficking is crucial for both normal cellular physiology and cell-cell communication. The conventional secretory route follows transport from the Endoplasmic reticulum (ER) to the plasma membrane via the Golgi apparatus. Alternative modes of secretion which can bypass the need for passage through the Golgi apparatus have been collectively termed as Unconventional protein secretion (UPS). UPS can comprise of cargo without a signal peptide or proteins which escape the Golgi in spite of entering the ER. UPS has been classified further depending on the mode of transport. Type I and Type II unconventional secretion are non-vesicular and non-SNARE protein dependent whereas Type III and Type IV dependent on vesicles and on SNARE proteins. In this review, we focus on the Type III UPS which involves the import of cytoplasmic proteins in membrane carriers of autophagosomal/endosomal origin and release in the extracellular space following SNARE-dependent intracellular membrane fusion. We discuss the role of vesicular SNAREs with a strong focus on VAMP7, a vesicular SNARE involved in exosome, lysosome and autophagy mediated secretion. We further extend our discussion to the role of unconventional secretion in health and disease with emphasis on cancer and neurodegeneration.

Macrophage migration into injured or infected tissue is a key aspect in the pathophysiology of many diseases where inflammation is a driving factor. Membrane‐type‐1 matrix metalloproteinase (MT1‐MMP) cleaves extracellular matrix components to facilitate invasion. Here we show that, unlike the constitutive MT1‐MMP surface recycling seen in cancer cells, unactivated macrophages express low levels of MT1‐MMP. Upon lipopolysaccharide (LPS) activation, MT1‐MMP synthesis dramatically increases 10‐fold at the surface by 15 hours. MT1‐MMP is trafficked from the Golgi complex to the surface via late endosomes/lysosomes in a pathway regulated by the late endosome/lysosome R‐SNAREs VAMP7 and VAMP8. These form two separate complexes with the surface Q‐SNARE complex Stx4/SNAP23 to regulate MT1‐MMP delivery to the plasma membrane. Loss of either one of these SNAREs leads to a reduction in surface MT1‐MMP, gelatinase activity and reduced invasion. Thus, inhibiting MT1‐MMP transport through this pathway could reduce macrophage migration and the resulting inflammation. [ABSTRACT FROM AUTHOR]

VAMP7 (vesicle-associated membrane protein) belongs to the intracellular membrane fusion SNARE (Soluble N-ethylmaleimide-sensitive factor attachment protein receptors) protein family. In this study, we used CRISPR/Cas9 genome editing technology to generate VAMP7 knockout (KO) human HeLa cells and mouse KO brain extracts in order to test the specificity and the background of a set of commercially available and homemade anti-VAMP7 antibodies. We propose a simple profiling method to analyze western blotting and use visual scoring for immunocytochemistry staining to determine the extent of the antibodies’ specificity. Thus, we were able to rank the performance of a set of available antibodies and further showed an optimized procedure for VAMP7 immunoprecipitation, which we validated using wild-type and KO mouse brain extracts.

VAMP7 (vesicle-associated membrane protein) belongs to the intracellular membrane fusion SNARE (Soluble N-ethylmaleimide-sensitive factor attachment protein receptors) protein family. In this study, we used CRISPR/Cas9 genome editing technology to generate VAMP7 knockout (KO) human HeLa cells and mouse KO brain extracts in order to test the specificity and the background of a set of commercially available and homemade anti-VAMP7 antibodies. We propose a simple profiling method to analyze western blotting and immunocytochemistry staining profiles and determine the extent of the antibodies’ specificity. Using this method, we were able to rank the performance of a set of available antibodies and further showed an optimized procedure for VAMP7 immunoprecipitation, which we validated using wild-type and KO mouse brain extracts.

The Arf4--rhodopsin complex (mediated by the VxPx motif in rhodopsin) initiates expansion of vertebrate rod photoreceptor cilia-derived light-sensing organelles through stepwise assembly of a conserved trafficking network. Here, we examine its role in the sorting of VAMP7 (also known as TI-VAMP) -- an R-SNARE possessing a regulatory longin domain (LD) -- into rhodopsin transport carriers (RTCs). During RTC formation and trafficking, VAMP7 colocalizes with the ciliary cargo rhodopsin and interacts with the Rab11--Rabin8--Rab8 trafficking module. Rab11 and Rab8 bind the VAMP7 LD, whereas Rabin8 (also known as RAB3IP) interacts with the SNARE domain. The Arf/Rab11 effector FIP3 (also known as RAB11FIP3) regulates VAMP7 access to Rab11. At the ciliary base, VAMP7 forms a complex with the cognate SNAREs syntaxin 3 and SNAP-25. When expressed in transgenic animals, a GFP--VAMP7ΔLD fusion protein and a Y45E phosphomimetic mutant colocalize with endogenous VAMP7. The GFP--VAMP7-R150E mutant displays considerable localization defects that imply an important role of the R-SNARE motif in intracellular trafficking, rather than cognate SNARE pairing. Our study defines the link between VAMP7 and the ciliary targeting nexus that is conserved across diverse cell types, and contributes to general understanding of how functional Arf and Rab networks assemble SNAREs in membrane trafficking. [ABSTRACT FROM AUTHOR]

Objectives: Five proteins from the soluble N-ethylmaleimide-sensitive factor activating protein receptor (SNARE) complex family were studied in normal hematopoietic cells in bone marrow; normal lymphocytes at different stages of maturation and differentiation in bone marrow, thymus, tonsil, and lymph node; malignant lymphomas; and leukemias.Methods: Sixty-eight reactive and 380 hematopoietic and lymphoid neoplasms were immunohistochemically stained for syntaxin 7 (STX7), vesicle-associated membrane proteins (VAMP2, VAMP7, VAMP8), and synaptosomal-associated protein 23 (SNAP23).Results: STX7 has potential for being a useful marker for distinguishing between normal B precursors (hematogones) vs B lymphoblasts, as well as between the "popcorn" cells of nodular lymphocyte-predominant Hodgkin lymphoma vs the Reed-Sternberg cells of classic Hodgkin lymphoma or the B cells of T-cell, histiocyte-rich B-cell lymphoma. VAMP2 is uniquely expressed by both reactive and malignant plasma cells, in contrast to B-cell non-Hodgkin lymphoma. There is differential expression of SNARE proteins in normal and neoplastic lymphoid tissue depending on lymphocyte maturation stage.Conclusions: Differential SNARE protein expression in the lymphoid system may have potential use in diagnosis and may offer clues to lymphoma biology. VAMP2 is a promising new plasma cell marker. [ABSTRACT FROM AUTHOR]

The neuropeptide VGF was recently proposed as a neurodegeneration biomarker. The Parkinson's disease-related protein leucine-rich repeat kinase 2 (LRRK2) regulates endolysosomal dynamics, a process that involves SNARE-mediated membrane fusion and could regulate secretion. Here we investigate potential biochemical and functional links between LRRK2 and v-SNAREs. We find that LRRK2 directly interacts with the v-SNAREs VAMP4 and VAMP7. Secretomics reveals VGF secretory defects in VAMP4 and VAMP7 knockout (KO) neuronal cells. In contrast, VAMP2 KO "regulated secretion-null" and ATG5 KO "autophagy-null" cells release more VGF. VGF is partially associated with extracellular vesicles and LAMP1+ endolysosomes. LRRK2 expression increases VGF perinuclear localization and impairs its secretion. Retention using selective hooks (RUSH) assays show that a pool of VGF traffics through VAMP4+ and VAMP7+ compartments, and LRRK2 expression delays its transport to the cell periphery. Overexpression of LRRK2 or VAMP7-longin domain impairs VGF peripheral localization in primary cultured neurons. Altogether, our results suggest that LRRK2 might regulate VGF secretion via interaction with VAMP4 and VAMP7. [Display omitted] • LRRK2 interacts with the endosomal v-SNAREs VAMP4 and VAMP7 • VAMP4 and VAMP7 mediate the secretion of the Parkinson's disease biomarker VGF • LRRK2 regulates the accumulation vs. secretion of VGF Filippini et al. report the direct interaction of leucine-rich repeat kinase 2 (LRRK2) with the endosomal vesicular SNAREs VAMP4 and VAMP7. LRRK2, VAMP4, and VAMP7 regulate the secretion of the Parkinson's disease biomarker VGF. They provide evidence for the role of LRRK2 in regulating unconventional secretion in the disease. [ABSTRACT FROM AUTHOR]

The growing pollen tube apex is dedicated to balancing exo- and endocytic processes to form a rapidly extending tube. As perturbation of either tends to cause a morphological phenotype, this system provides tractable model for studying these processes. Vesicle-associated membrane protein 7s (VAMP7s) are members of the soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) family that mediate cognate membrane fusion but their role in pollen tube growth has not been investigated. This manuscript identifies PiVAMP726 of Petunia inflata as a pollen-specific VAMP7 that localizes to the inverted cone of transport vesicles at the pollen tube tip. The endocytic marker FM4-64 was found to colocalize with yellow fluorescent protein (YFP)-PiVAMP726, which is consistent with PiVAMP726 containing an amino-acid motif implicated in endosomal localization, At high overexpression levels, YFP- PiVAMP726 inhibited growth and caused the formation of novel membrane compartments within the pollen tube tip. Functional dissection of PiVAMP726 implicated the N-terminal longin domain in negative regulation of the SNARE activity, but not localization of PiVAMP726. Expression of the constitutively active C-terminal SNARE domain alone, in pollen tubes, generated similar phenotypes to the full-length protein, but the truncated domain was more potent than the wild-type protein at both inhibiting growth and forming the novel membrane compartments. Both endo- and exocytic markers localized to these compartments in addition to YFP-PiVAMP726, leading to the speculation that PiVAMP726 might be involved in the recycling of endocytic vesicles in tip growth. [ABSTRACT FROM PUBLISHER]

Syntaxin 16, a Qa-SNARE (soluble N-ethylmaleimide-sensitive factor activating protein receptor), is involved in a number of membrane-trafficking activities, particularly transport processes at the trans-Golgi network (TGN). Recent works have now implicated syntaxin 16 in the autophagy process. In fact, syntaxin 16 appears to have dual roles, firstly in facilitating the transport of ATG9a-containing vesicles to growing autophagosomes, and secondly in autolysosome formation. The former involves a putative SNARE complex between syntaxin 16, VAMP7 and SNAP-47. The latter occurs via syntaxin 16's recruitment by Atg8/LC3/GABARAP family proteins to autophagosomes and endo-lysosomes, where syntaxin 16 may act in a manner that bears functional redundancy with the canonical autophagosome Qa-SNARE syntaxin 17. Here, I discuss these recent findings and speculate on the mechanistic aspects of syntaxin 16's newly found role in autophagy. [ABSTRACT FROM AUTHOR]