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Yeast dynamin, Vacuolar Protein Sorting 1 (Vps1), has been implicated in recycling traffic from the endosome to the trans-Golgi network (TGN). Previous research showed a genetic interaction of Vps1 with all components of the GARP tethering complex, which anchors vesicles at the late Golgi membrane. We used the yeast two-hybrid system and have identified a 33 amino acid segment of Vps51, a GARP subunit, that interacts with Vps1. Based on sequence homology between Vps51 and its mammalian homolog Ang2 in the 33 amino acids stretch, we identified two key residues of Vps51, E127 and Y129, that bind Vps1. The replacement of these residues led to severe defects in endosome-to-TGN transport of Snc1, providing evidence of the physiological relevance of the interaction of Vps51 with Vps1 for the traffic. Furthermore, our functional analysis revealed that Vps1 acts upstream of Vps51 and that the absence of Vps1 resulted in reduced localization levels of Vps51 and its binding partner Tlg1 to the late Golgi. Taken together, we propose that Vps1 functions with the GARP tethering machinery for efficient tethering/fusion at the TGN.

Protein recycling is an important cellular process required for cell homeostasis. Results from prior studies have shown that Vps1, a dynamin homologue in yeast, is implicated in protein recycling from the endosome to the trans-Golgi Network (TGN). However, the function of Vps1 in relation to Ypt6, a master GTPase in the recycling pathway, remains unknown. The present study reveals that Vps1 physically interacts with Ypt6 if at least one of them is full-length. We found that overexpression of full-length Vps1, but not GTP hydrolysis-defective Vps1 mutants, is sufficient to rescue abnormal phenotypes of Snc1 distribution provoked by loss of Ypt6, and vice versa. This suggests that Vps1 and Ypt6 function in parallel pathways instead of in a sequential pathway, and that GTP binding/hydrolysis of Vps1 is required for proper traffic of Snc1 toward the TGN. Additionally, we identified two novel Vps1 binding partners, Vti1 and Snc2, which function for the endosome-derived vesicle fusion at the TGN. Taken together, the present study demonstrates that Vps1 plays a role in later stages of the endosome-to-TGN traffic.