Revealing the architecture and dynamics of protein complexes at vacuolar membrane contact sites

• Applicants: Dr. Ayelen Gonzalez Montoro; Professorin Dr. Fan Liu
• Subject Area: Biochemistry ; Cell Biology
• Term: since 2020
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 453234580

Membrane contact sites (MCSs) are specialized domains where the membranes of two organelles are tethered in close apposition. They serve as platforms that enable the exchange of molecules and can influence organelle dynamics, hence playing a major role in coordinating metabolism in the cell. Despite its significant importance, the establishment, composition, and functionality of many MCSs remains partially or completely undescribed. In this proposal we set our focus on vacuolar MCSs. The vacuole of Saccharomyces cerevisiae recycles material from the endocytic and autophagic pathway to produce nutrients and building blocks for the cell. In addition, it critically contributes to cellular signaling as well as storage and regulation of the cytosolic concentration of numerous small molecules. We plan to use cross-linking mass spectrometry (XL-MS) as our major discovery tool to elucidate the molecular architecture and dynamics of protein complexes of the MCSs formed by the vacuole. To achieve a comprehensive interactome of the vacuolar membrane, we will advance the existing XL-MS method, by establishing a workflow incorporating a novel MS-cleavable and enrichable cross-linker. We will additionally enhance the detection of MCSs proteins by using overexpression strains known to extend specific contact sites. To unravel the functionality of the novel identified components and of the MCSs, we will characterize them with an array of complementary approaches such as fluorescence microscopy, biochemistry, genetics, and structural biology. Finally, by comparing interactomes from vacuoles isolated from cells grown under different conditions, we will assess the re-arrangements of the machinery in response to changes in metabolism. These studies will fill a crucial knowledge gap in MCS research and provide important insights into the molecular architecture and dynamics of vacuolar MCSs.

• DFG Programme: Research Grants