Nia J. Bryant

Regulated transport of the glucose transporter GLUT4

In muscle and fat cells, insulin stimulates the delivery of the glucose transporter GLUT4 from an intracellular location to the cell surface, where it facilitates the reduction of plasma glucose levels. Understanding the molecular mechanisms that mediate this translocation event involves integrating our knowledge of two fundamental processes — the signal transduction pathways that are triggered when insulin binds to its receptor and the membrane transport events that need to be modified to divert GLUT4 from intracellular storage to an active plasma membrane shuttle service.

Localization of phosphatidylinositol 3-phosphate in yeast and mammalian cells.

Phosphatidylinositol 3‐kinase (PI3K) regulates several vital cellular processes, including signal transduction and membrane trafficking. In order to study the intracellular localization of the PI3K product, phosphatidylinositol 3‐phosphate [PI(3)P], we constructed a probe consisting of two PI(3)P‐binding FYVE domains. The probe was found to bind specifically, and with high affinity, to PI(3)P both in vitro and in vivo. When expressed in fibroblasts, a tagged probe localized to endosomes, as detected by fluorescence microscopy. Electron microscopy of untransfected fibroblasts showed that PI(3)P is highly enriched on early endosomes and in the internal vesicles of multivesicular endosomes. While yeast cells deficient in PI3K activity (vps15 and vps34 mutants) were not labelled, PI(3)P was found on intralumenal vesicles of endosomes and vacuoles of wild‐type yeast. vps27Δ yeast cells, which have impaired endosome to vacuole trafficking, showed a decreased vacuolar labelling and increased endosome labelling. Thus PI(3)P follows a conserved intralumenal degradation pathway, and its generation, accessibility and turnover are likely to play a crucial role in defining the early endosome and the subsequent steps leading to multivesicular endosome formation.

Vps45p stabilizes the syntaxin homologue Tlg2p and positively regulates SNARE complex formation.

Sec1p‐like/Munc‐18 (SM) proteins bind to t‐SNAREs and inhibit ternary complex formation. Paradoxically, the absence of SM proteins does not result in constitutive membrane fusion. Here, we show that in yeast cells lacking the SM protein Vps45p, the t‐SNARE Tlg2p is down‐regulated, to undetectable levels, by rapid proteasomal degradation. In the absence of Vps45p, Tlg2p can be stabilized through abolition of proteasome activity. Surprisingly, the stabilized Tlg2p was targeted to the correct intracellular location. However, the stabilized Tlg2p is non‐functional and unable to bind its cognate SNARE binding partners, Tlg1p and Vti1p, in the absence of Vps45p. A truncation mutant lacking the first 230 residues of Tlg2p no longer bound Vps45p but was able to form complexes with Tlg1p and Vti1p in the absence of the SM protein. These data provide us with two valuable insights into the function of SM proteins. First, SM proteins act as chaperone‐like molecules for their cognate t‐SNAREs. Secondly, SM proteins play an essential role in the activation process allowing their cognate t‐SNARE to participate in ternary complex formation.

The t-SNARE Syntaxin 4 Is Regulated during Macrophage Activation to Function in Membrane Traffic and Cytokine Secretion

Activation of macrophages with lipopolysaccharide (LPS) induces the rapid synthesis and secretion of proinflammatory cytokines, such as tumor necrosis factor (TNFalpha), for priming the immune response. TNFalpha plays a key role in inflammatory disease; yet, little is known of the intracellular trafficking events leading to its secretion. In order to identify molecules involved in this secretory pathway, we asked whether any of the known trafficking proteins are regulated by LPS. We found that the levels of SNARE proteins were rapidly and significantly up- or downregulated during macrophage activation. A subset of t-SNAREs (Syntaxin 4/SNAP23/Munc18c) known to control regulated exocytosis in other cell types was substantially increased by LPS in a temporal pattern coinciding with peak TNFalpha secretion. Syntaxin 4 formed a complex with Munc18c at the cell surface of macrophages. Functional studies involving the introduction of Syntaxin 4 cDNA or peptides into macrophages implicate this t-SNARE in a rate-limiting step of TNFalpha secretion and in membrane ruffling during macrophage activation. We conclude that, in macrophages, SNAREs are regulated in order to accommodate the rapid onset of cytokine secretion and for membrane traffic associated with the phenotypic changes of immune activation. This represents a novel regulatory role for SNAREs in regulated secretion and in macrophage-mediated host defense.

Syntaxin 7 Complexes with Mouse Vps10p Tail Interactor 1b, Syntaxin 6, Vesicle-associated Membrane Protein (VAMP)8, and VAMP7 in B16 Melanoma Cells

Syntaxin 7 is a mammalian target solubleN-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) involved in membrane transport between late endosomes and lysosomes. The aim of the present study was to use immunoaffinity techniques to identify proteins that interact with Syntaxin 7. We reasoned that this would be facilitated by the use of cells producing high levels of Syntaxin 7. Screening of a large number of tissues and cell lines revealed that Syntaxin 7 is expressed at very high levels in B16 melanoma cells. Moreover, the expression of Syntaxin 7 increased in these cells as they underwent melanogenesis. From a large scale Syntaxin 7 immunoprecipitation, we have identified six polypeptides using a combination of electrospray mass spectrometry and immunoblotting. These polypeptides corresponded to Syntaxin 7, Syntaxin 6, mouse Vps10p tail interactor 1b (mVti1b), α-synaptosome-associated protein (SNAP), vesicle-associated membrane protein (VAMP)8, VAMP7, and the protein phosphatase 1M regulatory subunit. We also observed partial colocalization between Syntaxin 6 and Syntaxin 7, between Syntaxin 6 and mVti1b, but not between Syntaxin 6 and the early endosomal t-SNARE Syntaxin 13. Based on these and data reported previously, we propose that Syntaxin 7/mVti1b/Syntaxin 6 may form discrete SNARE complexes with either VAMP7 or VAMP8 to regulate fusion events within the late endosomal pathway and that these events may play a critical role in melanogenesis.

Traffic into the prevacuolar/endosomal compartment of Saccharomyces cerevisiae: A VPS45-dependent intracellular route and a VPS45-independent, endocytic route

The vps (vacuolar protein sorting) mutants have been used to dissect and characterize the vacuolar biogenesis pathway in the yeast Saccharomyces cerevisiae. The vps mutants were isolated through their loss of ability to correctly sort the vacuolar hydrolase CPY, which travels from Golgi membranes to the vacuole through a prevacuolar compartment. Over 50 VPS genes have been divided into 6 classes according to vacuolar morphology. Mutations in any one of the class E VPS genes, such as VPS27, lead to an exaggerated form of the prevacuolar compartment. This class E compartment contains endocytosed proteins as well as proteins en route to the vacuole, and is thus taken to represent an intersection point between the endocytic and biosynthetic pathways. Mutations in the class D gene VPS45 can be used to define a second transport intermediate along the vacuolar biogenesis pathway, Golgi-derived transport vesicles carrying vacuolar membrane proteins on their way to the vacuole. Here we demonstrate that the Sec1p-like protein Vps45p is required for the fusion of Golgi-derived vesicles with the prevacuolar compartment indicating that VPS45 functions before VPS27 in the vacuolar biogenesis pathway. In addition, we show that VPS45 function is not required for the delivery of endocytosed proteins to the prevacuolar compartment from the plasma membrane suggesting that the function of Vps45p is restricted to a single vesicular pathway.