Identification of the Client-binding Site on Vps74 and Its Role in Golgi Membrane Targeting

Abstract

The Golgi apparatus relies on precise sorting and recycling mechanisms to maintain its repertoire of resident membrane proteins, which in turn ensures accurate glycosylation of proteins and lipids. In yeast, the adaptor protein Vps74 is central to this process, linking many Golgi-resident enzymes (it’s clients) to the COPI-mediated retrograde pathway. However, the molecular determinants underlying how Vps74 binds its clients have remained poorly understood.

This work demonstrates that Vps74’s clients are sufficient to recruit Vps74 to Golgi membranes, revealing a novel mechanism of adaptor localization in addition to PI4P binding. These results extend our understanding of the Vps74-membrane recruitment by identifying the phosphatidylinositol-4-phosphate (PI4P) as a stabilizing co-factor, which functions particularly under low-client conditions. Importantly, the client-binding site of Vps74 was mapped to a membrane-opposing surface and is composed of the PI4P-binding pocket, a β-hairpin, and two evolutionarily conserved loops. These regions not only mediate recognition of Vps74 client tails, but can also engage two other Vps74’s binding partners: Arf1-GTP and the PI4P phosphatase Sac1.

Together, the findings reported in this thesis provide a framework for understanding how Vps74 regulates retention of resident integral membrane proteins across Golgi cisternae. They also suggest mechanisms by which client binding and lipid interactions are balanced against potential negative regulation by Sac1 and competition with Arf1. By elucidating the molecular basis of Vps74 function, this work advances our knowledge of Golgi homeostasis and offers insights into the conserved principles that underlie the function of its human homolog GOLPH3, a protein with established oncogenic significance. Therefore, results of this study have a translational potential, and could guide the search for the novel anti-cancer therapeutics.

Date of Award	2025
Original language	English
Awarding Institution	The Hong Kong University of Science and Technology
Supervisor	David Karl BANFIELD (Supervisor)