A Functional Role for VAP‐33 in Insulin‐Stimulated GLUT4 Traffic

Abstract

Soluble N-ethylmaleimide sensitive factor attachment protein receptors (SNAREs) are critical proteins in membrane fusion, in both regulated and constitutive vesicular traffic. In addition, proteins that interact with the SNAREs are thought to regulate fusion. Vesicle-associated membrane protein-2 (VAMP-2) is a SNARE protein involved in insulin-dependent glucose transporter 4 (GLUT4) traffic. VAMP-2 is required for productive GLUT4 incorporation into the plasma membrane. VAMPassociated protein of 33 kDa (VAP-33) is an integral membrane protein that binds VAMPs in vitro, and is hypothesized to be a regulator of VAMPs. In L6 skeletal myoblasts, which display insulin-dependent traffic of GLUT4, we show that VAP-33 colocalized significantly with VAMP-2 using indirect confocal immunofluorescence and biochemical cosegregation. Overexpression of wild-type VAP-33 in L6 myoblasts attenuated the insulindependent incorporation of myc-tagged GLUT4 into the plasma membrane, and this response was restored by co-overexpression of VAMP-2 linked to green fluorescent protein. Antibodies to VAP-33 microinjected into 3T3-L1 adipocytes abrogated the insulin-stimulated translocation of GLUT4 to the plasma membrane, as measured in adhered plasma membrane lawns. Immunopurified VAMP-2-containing compartments from L6 myotubes and 3T3-L1 adipocytes showed significant levels of VAP-33. We propose that VAP-33 may be a regulator of VAMP-2 availability for GLUT4 traffic and other vesicle fusion events. Key words: Endosome, exocytosis, SNARE, VAMP-2, vesicle traffic Received 24 January 2000, revised and accepted for publication 16 March 2000Soluble N-ethylmaleimide sensitive factor attachment protein receptor (SNARE) proteins have been the focus of multiple studies into vesicle-target membrane fusion. Syntaxin and synaptosome-associated protein of 25 kDa (SNAP-25) in one membrane and vesicle-associated membrane protein (VAMP) in an opposing membrane are thought to coalesce to form a higher order coiled-coil complex that mediates fusion (1). Primary, secondary, tertiary and quaternary structural features of SNAREs are conserved from yeast to mammals (2 -5) and the energy released during the formation of these quaternary coiled-coil structures has been proposed to contribute to vesicle fusion (1). Reconstitution experiments have demonstrated that SNAREs suffice for fusion in vitro (6). Multiple isoforms of each SNARE have been identified, each having a distinct tissue and subcellular distribution (for a comprehensive review of SNARE isoforms see (7)).In addition, specific proteins bind to individual SNARE molecules and are therefore considered to be potential SNARE regulators. Two of these SNARE-binding proteins, munc-18c and syntaxin interacting protein (synip), interact selectively with syntaxin-4 in 3T3-L1 adipocytes, and the extent of these interactions is regulated by insulin (8 -11). A protein interacting with the SNARE VAMP was also identified in Aplysia californica and called VAMP-associated protein of 33 kDa (VAP-33) (12). Antibodies to ...

A Highly Conserved Binding Site in Vesicle-associated Membrane Protein-associated Protein (VAP) for the FFAT Motif of Lipid-binding Proteins

“…This altered targeting mapped to the transmembrane domain and may be associated with the ability of this region of most VAPs to dimerize, as they contain a GXXXG motif, whereas it is unlikely that the transmembrane domain of Scs2 dimerizes (17,38). The exit of human VAP-A from the yeast ER is consistent with previous reports of fly and vertebrate VAPs outside of the ER (9,11,29,30). The traffic of human VAP-A to the yeast vacuole, once it has escaped from the ER, may result from recognition of its transmembrane domain by a default degradation pathway (39).…”

“…Only chimeras containing the MSP-VAP domain from Scs2 rescued well, whereas this domain from human VAP-A functioned poorly, indicating an additional function of the MSP-VAP domain of Scs2. One possibility is that this additional function relates to intracellular targeting, because human VAP-A has been reported to localize to membrane compartments outside of the ER (9,11,29,30). We therefore examined the intracellular targeting of human VAP-A and found it not only in the ER but also in the vacuole (Fig.…”

Differential Regulation of Endoplasmic Reticulum Structure through VAP-Nir Protein Interaction

“…It belongs to a highly conserved family of proteins, which are implicated in the regulation of neurotransmitter release, ER-Golgi and intra-Golgi transport, Glu4 (glucose transporter 4) trafficking, stabilization of presynaptic microtubules, and the expression of phospholipid biosynthetic genes (19,(21)(22)(23)(24)(25). These diverse functions have been demonstrated in different species and cell types, and are mediated by different members of this family.…”

Protein Phosphatase 2Cϵ Is an Endoplasmic Reticulum Integral Membrane Protein That Dephosphorylates the Ceramide Transport Protein CERT to Enhance Its Association with Organelle Membranes

“…VAPA is composed of two conserved domains, an amino-terminal immunoglobulin-like ␤ sheet responsible for FFAT motif binding and a carboxyl-terminal transmembrane domain (8). In addition to its role in recruiting FFAT motif-targeted proteins to ER membranes, VAPA has been proposed to function in vesicle trafficking (1,(12)(13)(14), in the organization of the microtubule network (10, 15), and in the replication of hepatitis C virus RNA (16,17). In mammalian cells ceramide is synthesized in the ER and transported to the Golgi apparatus where it is converted to sphingomyelin (SM).…”