RESUMEN
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.
Asunto(s)
Dinaminas/metabolismo , Endosomas/metabolismo , Proteínas de Unión al GTP/metabolismo , Aparato de Golgi/metabolismo , Proteínas R-SNARE/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Transporte Vesicular/metabolismo , Vesículas Citoplasmáticas/genética , Vesículas Citoplasmáticas/metabolismo , Dinaminas/genética , Endosomas/genética , Proteínas de Unión al GTP/genética , Aparato de Golgi/genética , Unión Proteica , Transporte de Proteínas/genética , Proteínas R-SNARE/genética , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Transporte Vesicular/genética , Red trans-Golgi/genética , Red trans-Golgi/metabolismoRESUMEN
Retrograde passage of a transport carrier entails cargo sorting at the endosome, generation of a cargo-laden carrier and its movement along cytoskeletal tracks towards trans-Golgi network (TGN), tethering at the TGN, and fusion with the Golgi membrane. Significant advances have been made in understanding this traffic system, revealing molecular requirements in each step and the functional connection between them as well as biomedical implication of the dysregulation of those important traffic factors. This review focuses on describing up-to-date action mechanisms for retrograde transport from the endosomal system to the TGN.
Asunto(s)
Endosomas/metabolismo , Red trans-Golgi/metabolismo , HumanosRESUMEN
The yeast dynamin Vps1 acts cooperatively with many proteins at diverse cellular locations for endocytosis, protein sorting, and membrane fusion and fission. It has been proposed that Vps1 is functionally linked to clathrin heavy chain 1 (Chc1), but the question of how, where, and when they function together remains unknown. Here we report that Vps1 arrives at the Golgi after clathrin, and that loss of Vps1 leads to a shift in the cellular localization of clathrin to the late endosome and vacuole, not vice versa. Our two-hybrid-based approach provides evidence that full-length Vps1 and its truncated versions bind to the C-terminal region of the Chc1. Cells lacking both Vps1 and Chc1 displayed more severe defects in carboxypeptidase Y (CPY) sorting at the Golgi than those in Vps1-deficient cells. Further, these Vps1 fragments became dominant-negative for CPY sorting upon overexpression. These results suggest that Vps1 binds to Chc1 and functions together at the Golgi for efficient Golgi-to-endosome membrane trafficking. In addition, we found that Vps1, without the aid of clathrin, plays a role in controlling the number and turnover of late Golgi.
Asunto(s)
Clatrina/metabolismo , Dinaminas/metabolismo , Proteínas de Unión al GTP/metabolismo , Aparato de Golgi/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Transporte Vesicular/metabolismo , Proteínas de Unión al GTP/genética , Homeostasis , Saccharomyces cerevisiae/genética , Proteínas de Transporte Vesicular/genéticaRESUMEN
The dynamin-like protein, Vps1, is a GTPase involved in cargo sorting and membrane remodeling in multiple cellular trafficking pathways. Recently, Vps1 has been shown to genetically interact with ESCRT subunits. We tested the hypothesis that the functional connection of Vps1 with some of these subunits of ESCRT complexes occurs via a physical interaction. By utilizing the yeast two-hybrid system, we revealed that Vps1 physically interacts with the ESCRT-II subunits, Vps22 and Vps36, and the ESCRT-III subunit Vps24. We found that Vps1 and ESCRT-II components colocalize with Pep12, an endosomal marker. Additionally, loss of Vps1 or depletion of the GTPase activity of Vps1 results in a moderate defect in Cps1 targeting to the vacuole. Here, we discussed the potential implications of Vps1 and ESCRT interaction and their roles in the endosome-to-vacuole traffic. In summary, yeast dynamin interacts with ESCRT II and III complexes, and it functions in Cps1 trafficking toward the vacuole.