Mechanism of HOPS-dependent AP-3 vesicles targeting to the yeast vacuole

• Applicant: Professor Dr. Christian Ungermann
• Subject Area: Biochemistry ; Cell Biology
• Term: since 2019
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 422067766

The AP-3 pathway delivers several key proteins to the vacuole, which determine its identity and function as the terminal organelle of the endolysosomal pathway. Among these are the Yck3 casein kinase and the SNAREs Vam3 and Nyv1. Yck3 in turn phosphorylates several proteins involved in the fusion cascade, presumably at the vacuole (Fig. 1B). Yck3 resides both in vesicles and on the vacuole, and its loss also strongly impairs the AP-3 transport. It thus remains entirely unclear, where and how Yck3 function is needed. The project therefore aims to understand the molecular mechanism of how Yck3 regulates the AP-3 pathway, and thus clarify the organization of the underlying machinery required for AP-3 fusion with the vacuole (or endosome). In Aim 1, we will ask which specific part of the fusion machinery is needed on AP-3 vesicles. We will therefore establish a novel protocol to purify native AP-3 vesicles using a specific AP-3 cargo as a bait and determine the AP-3 specific fusion machinery using organelle purification protocols and mass spectrometry. In Aim 2, we will clarify, on which organelle membrane Yck3 is required for efficient AP-3 transport to the vacuole and how AP-3 transport is regulated. We will design a series of Yck3 constructs, which will trap Yck3 at endosomes or the vacuole. This will clarify, where cargo phosphorylation is most likely required. In this context, we will determine, how Yck3 phosphorylation (which occurs mostly at the C-terminal part) affects its function, and which kinase is responsible for this. In Aim 3, we will finally use purified AP-3 vesicles and our reconstituted system to recapitulate the fusion of AP-3 vesicles with the vacuole. Based on our findings in Aim 1 and Aim 2, we will establish an heterotypic fusion assay using purified organelles or reconstituted proteoliposomes and liposomes mimicking the vacuole to test the requirement of the involved fusion machinery (SNAREs, HOPS, Ypt7) and regulators (Mon1-Ccz1, Yck3) in this assay. Possible mutants in each protein will be used as a control. The combined approaches should clarify, how AP-3 vesicles are specifically targeted to the vacuole surface and determine its identity.

• DFG Programme: Research Grants