SNARE proteins have been proposed to mediate all intracellular membrane fusion events. There are over 30 SNARE family members in mammalian cells and each is found in a distinct subcellular compartment. It is likely that SNAREs encode aspects of membrane transport specificity but the mechanism by which this specificity is achieved remains controversial. Functional studies have provided exciting insights into how SNARE proteins interact with each other to generate the driving force needed to fuse lipid bilayers.

SNARE-mediated membrane fusion.

Insulin stimulates the transport of glucose into fat and muscle cells. Although the precise molecular mechanisms involved in this process remain uncertain, insulin initiates its actions by binding to its tyrosine kinase receptor, leading to the phosphorylation of intracellular substrates. One such substrate is the Cbl proto-oncogene product. Cbl is recruited to the insulin receptor by interaction with the adapter protein CAP, through one of three adjacent SH3 domains in the carboxy terminus of CAP. Upon phosphorylation of Cbl, the CAP-Cbl complex dissociates from the insulin receptor and moves to a caveolin-enriched, triton-insoluble membrane fraction. Here, to identify a molecular mechanism underlying this subcellular redistribution, we screened a yeast two-hybrid library using the amino-terminal region of CAP and identified the caveolar protein flotillin. Flotillin forms a ternary complex with CAP and Cbl, directing the localization of the CAP-Cbl complex to a lipid raft subdomain of the plasma membrane. Expression of the N-terminal domain of CAP in 3T3-L1 adipocytes blocks the stimulation of glucose transport by insulin, without affecting signalling events that depend on phosphatidylinositol-3-OH kinase. Thus, localization of the Cbl-CAP complex to lipid rafts generates a pathway that is crucial in the regulation of glucose uptake.

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CAP defines a second signalling pathway required for insulin-stimulated glucose transport

Ceulemans, Hugo, and Mathieu Bollen. Functional Diversity of Protein Phosphatase-1, a Cellular Economizer and Reset Button. Physiol Rev 84: 1–39, 2004; 10.1152/physrev.00013.2003.—The protein serin...

https://www.physiology.org/doi/pdf/10.1152/physrev.00013.2003

Functional Diversity of Protein Phosphatase-1, a Cellular Economizer and Reset Button

The release of neurotransmitters by Ca2+-triggered synaptic vesicle exocytosis is an exquisitely regulated process that is fundamental for interneuronal communication. This process involves several steps and is controlled by a protein machinery that must prevent release before Ca2+ entry into presynaptic terminals, and yet must rapidly induce release on Ca2+ influx. Extensive studies of the components of this machinery have indicated that SNAREs and Munc18-1 are central proteins for membrane fusion during exocytosis. An increasing amount of information derived from a convergence of structural, physiological and genetic studies is providing important insights into the mechanism of neurotransmitter release.

http://www.uniroma2.it/didattica/NEURO/deposito/SNARE.pdf

Snares and Munc18 in synaptic vesicle fusion.

SNAREs (soluble N-ethylmaleimide-sensitive factor attachment protein receptors) and Rab-GTPases, together with their cofactors, mediate the attachment step in the membrane fusion of vesicles. But how bilayer mixing--the subsequent core process of fusion--is catalysed remains unclear. Ca2+/calmodulin controls this terminal process in many intracellular fusion events. Here we identify V0, the membrane-integral sector of the vacuolar H+-ATPase, as a target of calmodulin on yeast vacuoles. Between docking and bilayer fusion, V0 sectors from opposing membranes form complexes. V0 trans-complex formation occurs downstream from trans-SNARE pairing, and depends on both the Rab-GTPase Ypt7 and calmodulin. The maintenance of existing complexes and completion of fusion are independent of trans-SNARE pairs. Reconstituted proteolipids form sealed channels, which can expand to form aqueous pores in a Ca2+/calmodulin-dependent fashion. V0 trans-complexes may therefore form a continuous, proteolipid-lined channel at the fusion site. We propose that radial expansion of such a protein pore may be a mechanism for intracellular membrane fusion.

Trans -complex formation by proteolipid channels in the terminal phase of membrane fusion

Intracellular membrane fusion is crucial for the biogenesis and maintenance of cellular compartments, for vesicular traffic between them, and for exo- and endocytosis. Parts of the molecular machinery underlying this process have been identified, but most of these components operate in mutual recognition of the membranes. Here it is shown that protein phosphatase 1 (PP1) is essential for bilayer mixing, the last step of membrane fusion. PP1 was also identified in a complex that contained calmodulin, the second known factor implicated in the regulation of bilayer mixing. The PP1-calmodulin complex was required at multiple sites of intracellular trafficking; hence, PP1 may be a general factor controlling membrane bilayer mixing.

http://science.sciencemag.org/content/sci/285/5430/1084.full.pdf

A. Glatz

Papers

Control of the terminal step of intracellular membrane fusion by protein phosphatase 1.