Functional homology of mammalian syntaxin 16 and yeast Tlg2p reveals a conserved regulatory mechanism

Abstract

Membrane fusion in all eukaryotic cells is regulated by the formation of specific SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) complexes. The molecular mechanisms that control this process are conserved through evolution and require several protein families, including Sec1p/Munc18 (SM) proteins. Here, we demonstrate that the mammalian SNARE protein syntaxin 16 (Sx16, also known as Syn16) is a functional homologue of the yeast SNARE Tlg2p, in that its expression fully complements the mutant phenotypes of tlg2Δ mutant yeast. We have used this functional homology to demonstrate that, as observed for Tlg2p, the function of Sx16 is regulated by the SM protein Vps45p. Furthermore, in vitro SNARE-complex assembly studies demonstrate that the N-terminal domain of Tlg2p is inhibitory to the formation of SNARE complexes, and that this inhibition can be lifted by the addition of purified Vps45p. By combining these cell-biological and biochemical analyses, we propose an evolutionarily conserved regulatory mechanism for Vps45p function. Our data support a model in which the SM protein is required to facilitate a switch of Tlg2p and Sx16 from a closed to an open conformation, thus allowing SNARE-complex assembly and membrane fusion to proceed.

The COG complex interacts with multiple Golgi SNAREs and enhances fusogenic SNARE complexes assembly

“…5A, Stx6 and Vti1a could bind the immobilized GST-Stx16 but not GST. The presence of Vps45 enhanced their interactions (by 143610% for Vti1a and by 223612.3% for Stx6), consistent with previous reports (Struthers et al, 2009). Remarkably, the addition of Cog4 subunit further enhanced the binding of Vti1a and Stx6 to Stx16 (by 24068.5% and 400615%, respectively), suggesting that the Cog4 subunit facilitates tSNARE complex assembly.…”

“…We could reconstitute the assembly of Stx6-Stx16-Vti1a in the absence or presence of either Vps45 or Cog4, and showed that both Cog4 and Vps45 enhance tSNARE complex assembly. While the stimulatory effect of Vps45 on SNARE complex assembly has been previously shown (Struthers et al, 2009), the influence of Cog4 on tSNARE assembly is shown here for the first time. The entire COG complex, which can bind directly the three tSNAREs, might have an even stronger effect on tSNAREs assembly.…”

The N-terminal peptide of the syntaxin Tlg2p modulates binding of its closed conformation to Vps45p

“…First, we previously showed that deletion of the first 230 residues of Tlg2p in vps45⌬ cells leads to endosomal SNARE complex formation (35), suggesting that in the absence of Vps45p, the closed conformation of Tlg2p inhibits SNARE complex assembly. Second, we have recently demonstrated that either removal of the Tlg2p H abc domain or the addition of Vps45p increases the rate of endosomal SNARE complex assembly in vitro (36). Here, we show that binding of Vps45p to the C-terminal binding site requires the H abc , linker and SNARE motif regions of Tlg2p.…”

Endosomal sorting of GLUT4 and Gap1 is conserved between yeast and insulin-sensitive cells

“…This study gapped pNB701 as described previously (Struthers et al, 2009). pHA-G4/Gap1, the retroviral expression vector encoding HA-tagged human GLUT4 with the entire C-terminal cytosolic tail (residues 470-509; please see supplementary material Table S1) replaced with the analogous region of the yeast protein Gap1 (residues 547-602; please see supplementary material Table S1) was constructed using SOEing PCR to generate a GLUT4/GAP1 chimeric ORF as follows.…”