Functional analysis of proteins involved in mitochondrial lipid homeostasis

• Applicant: Dr. Kai Stefan Dimmer
• Subject Area: Cell Biology ; Biochemistry
• Term: from 2014 to 2023
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 264118468

Mitochondria contain two distinct membranes that fulfill important functions and have each a unique composition of proteins and lipids. Whereas the biogenesis of mitochondrial proteins was studied intensively over the last decades, little is known about the trafficking and homeostasis of mitochondrial lipids. A major part of mitochondrial lipids have to be imported from other cellular compartments and only few lipids are synthesized inside the organelle from imported precursors. With the identification of ERMES, a protein complex at the heart of ER-mitochondria encounter structures, a first mediator of lipid flux in and out of mitochondria in S. cerevisiae (yeast) was identified. Yet, mitochondria are in contact with other organelles for example the vacuole, peroxisomes and lipid droplets (LDs). Several reports suggest that lipids can be transferred to mitochondria at these contact sites. While the contact to vacuole and peroxisomes were studied to some detail, only little is known about the molecular basis of tethers at the contact sites to lipid droplets. Furthermore, the transport proteins that bind and transfer lipids are largely unknown. Although mitochondrial membranes have the lowest sterol content of all organellar membranes, sterols are required for proper mitochondrial function. Ergosterol is synthesized in the ER and can be stored as sterolesters in lipid droplets. Hence, it has to be imported into mitochondria, yet the processes that determine mitochondrial sterol import and homeostasis are largely unknown. To shed new light on mitochondrial lipid homeostasis in yeast, the proposed project will address several fundamental questions: i) How do mitochondria interact with lipid droplets and what is the molecular identity of the involved tethers? ii) Which molecules are exchanged between the two organelles?, and iii) Which proteins are involved in mitochondrial sterol handling? We will combine yeast genetic approaches, fluorescence microscopy, protein biochemical assays, mass spectrometric analysis and lipidomics to shed light on these issues. Particularly, we will search by sub-cellular fractionations followed by mass spectrometry for proteins that tether LD to mitochondria and will study the influence of mitochondria on LDs and vice versa. Next, we will analyze the involvement of the ERMES complex and other reported ergosterol-binding proteins in sterol homeostasis of mitochondria. Furthermore, we will study the role of the three mitochondrial proteins Mcp1-3, which we recently identified, in ergosterol trafficking. The focus will lie on the conserved inner membrane protein Mcp2 that has an atypical kinase domain located in the intermembrane space (IMS). We will search for substrates of Mcp2 and will study its interactions with the only known mitochondrial triacylglycerol lipase, Tgl2. Overall, the project proposed will offer novel understandings of mitochondrial lipid homeostasis.

• DFG Programme: Research Grants