Structure-Function Studies of Regulators in Vesicle Trafficking.

摘要

In eukaryotic cells, proteins, lipids and nutrients are moved from one organelle to another via membrane-bound vesicles. Each step in transport is orchestrated by multiple proteins and is exquisitely regulated, including the delivery of vesicles to the correct destinations. Small GTPases, particularly those in the Rab family, and phosphoinositides in the membranes play critical roles in regulation and especially as determinants of membrane identity. Distinct Rabs and phosphoinositides localize to the membranes of different organelles. For my thesis work, I undertook two projects. The first project focuses on how Rab GTPases are activated by guanine nucleotide exchange factors (GEFs) in the DENN (differentially expressed in normal and neoplastic cells)-domain family, where Rab activation is prerequisite for their role as determinants of membrane identity. Specifically, I determined the crystal structure of the DENN-domain GEF DENND1BS in complex with its substrate Rab35. In combination with kinetic studies, the structure suggested how DENN-domains recognize and activate their substrates. The second project dissects the structure and function of the lipid kinase complex that is responsible for generating the phosphoinositide PI4P at the plasma membrane. PI4P is important for membrane trafficking as a marker of membrane identity; but additionally it is critical for signal transduction as a precursor for signaling molecules such as inositol trisphosphate, diacylglycerol, or PI(3,4,5)P3. In this work, I determined the structures of two regulatory components of the lipid kinase complex, Efr3 and Ypp1. Combining yeast genetics, cell biology, biochemical and biophysical methods, this work provides a detailed model of how the complex is assembled and targeted to the plasma membrane, providing valuable insights into PI4P regulation at the plasma membrane.