Munc18 Proteins

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Munc18 Proteins

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Munc18 Proteins Background

The Sec1/Munc18 (SM) Protein Family

The first family member of the Sec1/Munc18 (SM) proteins, unc18, was identified by Dr. Sydney Brenner more than three decades ago in a genetic screen of uncoordinated (unc) mutants in C. elegans. Five years after, the S. ceriviseae homolog Sec1 was isolated independently by Novick and Schekman through studies of a secretion-deficient mutant strain. The discovery of the mammalian unc18 (Munc18) then followed more than a decade after from the laboratory of Dr. Thomas Südhof, where this study was the first to identify that Munc18a is functionally coupled to the SNARE machinery and mediate fusion. To date, a total of seven mammalian SM members (Munc18a/b/c, Sly1, Vps45, Vps33a/b) have been identified, although only the Munc18 isoforms have been implicated in exocytosis. X-ray crystallographic study of SM-SNARE proteins identified these proteins in three species: first, from a complex formed between the murine neuronal SM protein, Munc18a, and the cytosolic domain of Syn-1A; second, from uncomplexed squid neuronal Sec1 (or s-Sec1); third, from a complex formed between the S. cerevisiae SM protein Sly1p and a short N-terminal peptide of the yeast Syntaxin Sed5p. From these models, the SM family is identified as a conserved set of 60-70kDa hydrophilic proteins, composed of a ~600 a.a. sequence, and has a characteristic property of folding three dimensionally into an arch-shaped molecule that is devoid of transmembrane segments.

The SM proteins are composed of three domains (1, 2 and 3), of which domain 3 in the mammalian Munc18a has been further subdivided into domains 3a and 3b, and where the domain 3a has been suggested to be the sub-domain to signal the release of Syn-1A from Munc18a upon effector signaling. Based on this mammalian model, the Syn-1A binding site is located between the central cavity formed between domains 1 and 3a of Munc18a, although the domain 1 of Munc18a has a greater number of contacts than domain 3a with the Syn-1A binding interface. Consistently, the s-Sec1 crystal complex reveals similar features on the domains, although a further hinge region between domains 1 and 2 in the s-Sec1 structure is required to release its cognate t-SNAREs.

Structure of Munc18a and Syntaxin-1A

Figure 1 Structure of Munc18a and Syntaxin-1A.

Munc18 proteins and the regulation of exocytosis

The earliest evidence in understanding the functions of Munc18 proteins in regulated exocytosis originated from early studies associating Munc18 as an inhibitor of SNARE-mediated fusion at some steps along the vesicle cycle. This idea was bolstered from over-expression studies conducted in the Drosophila melanogaster SM protein ROP and provided by the fact that in addition to sharing a 65% sequence homology with Munc18a, ROP inhibited neurotransmission and importantly the co-expression of Syn-1 reversed the inhibition. This supports the important concept that a 1:1 molar stoichiometry in Munc18-Syntaxin complexes is critical for neuroexocytosis, and raises the possibility that negative regulatory function of Munc18 proteins is present only when they are over-expressed beyond physiological levels.

Consistently, over-expression studies using biochemical strategies in various model systems indicate that Munc18 proteins inhibit vesicle cargo transport at distinct steps along the vesicle cycle. For instance, a study showed that increased level of Munc18a in pancreatic β-cell inhibits exocytosis by reducing the priming and refilling of vesicle pools, while over-expression of the related isoform Munc18b inhibits both apical exocytosis in Caco2 cells and capacitance release in chromaffin cell. Dr. Debbie Thurmond and colleagues have likewise reported that Munc18c prevents GLUT4 vesicle fusion in adipocytes and insulin secretion is markedly reduced in transgenic mice over-expressing Munc18c, in major part the result of a reduced second-phase secretion.

Consistently, a number of over-expression studies utilizing site-specific peptides support this plausible hypothesis of an inhibitory role of Munc18 proteins in SNARE-mediated exocytosis. expression of the Munc18b isoform, its entire domain 3 (a/b) from amino acids 246-488 that includes a Syntaxin-binding domain, or a specific “effector loop” that contains domain 3 from amino acids 433-488 were reported to specifically inhibit granule exocytosis in mast cells. In the adipocyte model, over-expression of Munc18c or application of a Munc18c “effector loop” peptide inhibits insulin-stimulated GLUT4 translocation to the PM. Likewise, peptides originating from either Munc18a or Munc18c restrict granule exocytosis in permeabilized human platelets.

A more inclusive perspective on this hypothesis came with noted exceptions among published reports challenging the inhibitory role of Munc18 proteins. In cell-lines, overexpressed Munc18a has no effect on secretion in chromaffin or PC12 cells or on insulin secretion in neuroendocrine INS-1 β-cell. Similarly, acute increases in the related levels of Munc18b in skeletal muscle cell or Munc18c in mast cell had no effect on exocytoses, respectively. The apparent confusing nature of Munc18 regulation in exocytosis was further evident when over-expression of Munc18a in primary chromaffin cells and motor neurons markedly increased the docking and fusion vesicles. Meanwhile, two Munc18c peptides modeled against a Syn-4 binding region were found to enhance Ca2+-stimulated platelet granule exocytosis. Therefore, a natural question that arises is what accounts for these discrepancies in Munc18 functions? One possible explanation could involve the evolutionary specialization of Munc18 isoforms to accommodate and orchestrate the exquisite trafficking steps in mammalian cells. A second reason could arise with the fact that the primary target of the 2 peptides designed by Houng et al. (2003) was different than those peptides used in previous studies. To date, all of the functional effects reported on Munc18 proteins do emphasize the importance that the molar stoichiometry of Munc18-Syntaxin species are important, and that either over-expression (as discussed) or under-expression may cause deleterious effects at some steps leading to membrane fusion. Consistent with this latter hypothesis, genetic deletion of Munc18a in mice are reported to be lethal.

Increasing studies now add to the hypothesis that Munc18 proteins are involved intimately in multiple functions along the stages of SG trafficking, in addition to their well-known regulation of Syntaxin configurations. First, Munc18a has been reported to directly stabilize and control the trafficking of Syn-1A to the PM, acting as a Syntaxin chaperone. Second, Munc18 proteins serve as a quintessential docking factor, in which the frequency of docking events was markedly reduced in a Munc18a knock-out mouse model and this could partially be Syntaxin-dependent. Third, Munc18a has been shown to enhance SNARE-mediated lipid fusion, where specifically, Munc18a facilitates the assembly of SNARE complexes between VAMP and co-expressed t-SNARE binary complexes. Fourth, Munc18a has been implicated to control fusion pore dynamics by modifying the kinetics of single-granule exocytotic release events, although there is recent data from the work of Gulyás-Kovács et al. (2007) that argues against this notion. Taken together, these studies above reinforce the over-arching hypothesis that Munc18 proteins can exert multiple functions throughout the process of exocytosis.

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