The Sec1/Munc18 Protein Vps45 Regulates Cellular Levels of Its SNARE Binding Partners Tlg2 and Snc2 in Saccharomyces cerevisiae

Abstract

Intracellular membrane trafficking pathways must be tightly regulated to ensure proper functioning of all eukaryotic cells. Central to membrane trafficking is the formation of specific SNARE (soluble N-ethylmeleimide-sensitive factor attachment protein receptor) complexes between proteins on opposing lipid bilayers. The Sec1/Munc18 (SM) family of proteins play an essential role in SNARE-mediated membrane fusion, and like the SNAREs are conserved through evolution from yeast to humans. The SM protein Vps45 is required for the formation of yeast endosomal SNARE complexes and is thus essential for traffic through the endosomal system. Here we report that, in addition to its role in regulating SNARE complex assembly, Vps45 regulates cellular levels of its SNARE binding partners: the syntaxin Tlg2 and the v-SNARE Snc2: Cells lacking Vps45 have reduced cellular levels of Tlg2 and Snc2; and elevation of Vps45 levels results in concomitant increases in the levels of both Tlg2 and Snc2. As well as regulating traffic through the endosomal system, the Snc v-SNAREs are also required for exocytosis. Unlike most vps mutants, cells lacking Vps45 display multiple growth phenotypes. Here we report that these can be reversed by selectively restoring Snc2 levels in vps45 mutant cells. Our data indicate that as well as functioning as part of the machinery that controls SNARE complex assembly, Vps45 also plays a key role in determining the levels of its cognate SNARE proteins; another key factor in regulation of membrane traffic.

Co-regulation of theGlycine maxsoluble N-ethylmaleimide-sensitive fusion protein attachment protein receptor (SNARE)-containing regulon occurs during defense to a root pathogen

“…The experiments presented here have also examined the relative changes in transcript abundance of SNARE, demonstrating that the genes appear to be co-regulated. The co-regulation of different vesicle components observed here in this system has been seen in other organisms, some of them non-plant systems, and functional genomics screens have revealed this co-regulation can be quite extensive (Shanks et al 2012;Liberali et al 2014;Pant et al 2014Pant et al , 2015aZick et al 2015). However, very little published data is available in plants.…”

The Thr224Asn mutation in the VPS45 gene is associated with the congenital neutropenia and primary myelofibrosis of infancy

“…[16][17][18][19][20] In yeast, loss of Vps45 function leads to the deceleration of cell growth rate, defective endosomal trafficking concomitant with reduced cellular levels of cognate SNARE proteins, increased sensitivity to osmotic stress, and accumulation of transport vesicles. 13,21 Similarly, the absence of Vps45 in Drosophila melanogaster blocks the fusion of endocytic vesicles into the endosome, resulting in vesicle accumulation and the absence of later endocytic structures. 19 Human Vps45 was previously shown to regulate vesicular trafficking between the trans-Golgi network and early endosomes and the recycling of plasma membrane receptors.…”

“…Yeast strains used were SEY6210 11 (containing endogenous Vps45) and a congenic vps45D (lacking endogenous Vps45) strain that was constructed by using pNOzY13 to disrupt VPS45, as described in Bryant and James. 12 Cellular levels of wild-type Vps45 and Vps45T238N expressed from pCOG070 and pMC007, and endogenous Tlg2 in the same cells, were quantified by densitometry using ImageJ software (National Institutes of Health) in relation to Pgk1 levels (which were used as a loading control) using immunoblot analysis as described in Shanks et al 13 …”

“…12 Loss of Vps45 function in yeast results in multiple phenotypes including reduced cellular levels of Tlg2. 13 To investigate the effect of the patients' mutation on Vps45 function, we introduced the analogous mutation, Thr238Asn, into the yeast protein and determined the cellular level of Tlg2. This analysis revealed that yeast cells expressing Vps45T238N from a plasmid in place of endogenous Vps45 have reduced cellular levels of Tlg2 by 44.5 6 9.4% (n 5 3) compared with cells expressing wild-type Vps45 under the same conditions ( Figure 3A).…”

Formation of Tubulovesicular Carriers from Endosomes and Their Fusion to the trans-Golgi Network

“…In addition, the Sec1/Munc18 (SM) protein VPS45 might regulate this fusion event. VPS45 is the only SM family member found at the Golgi in mammalian cells (Tellam et al, 1997) and in yeast mediates the assembly of an analogous SNARE complex (Bryant and James, 2001;Shanks et al, 2012) via physical interaction with Tlg2p, the functional homolog of Stx16 (Shanks et al, 2012).…”