Molecular mechanisms regulating protein transport and targeting
Eukaryotic cells compartmentalize biological functions in a series of membrane-bound organelles. The unique composition of each compartment is maintained despite the continuous movement of proteins and lipids within the cell. To achieve that proteins are specifically targeted to various subcellular compartments. Furthermore, regulated protein targeting also plays a key role in receptor recycling, cell motility and cytokinesis (cell division).
Cells achieve protein targeting through the use of transport vesicles equipped with complex arrays of proteins that regulate vesicle formation, transport, and fusion. Small Rab GTPases are the key proteins involved in membrane traffic. Rabs function as "address" tags of transport vesicles by recruiting various effector proteins to the membranes. Critical questions in understanding the roles of Rab proteins are the identity and specificity of these effectors. Since most Rab GTPases have multiple effector proteins, the mechanisms and regulation of their binding are essential for understanding of Rab functions in membrane traffic.
The identification of Rab binding proteins and understanding their function has been main focus of our laboratory in a last few years. The work in my laboratory led to identification of the novel family of Rab binding proteins, known as FIPs. Furthermore, we have shown that different Rab-FIP complexes may serve as "targeting patches", thus determining the fate of transport vesicle. Three main projects are being currently investigated in the laboratory: (1) structure/function analysis of Rab-FIP protein complexes; (2) the role of Rab-FIP complexes in regulating protein and membrane targeting; (3) identification of novel proteins interacting with Rab-FIP "targeting patches". To address these questions, several methods will be combined, including structural studies using circular dichroism, x-ray crystallography and NMR, immunoprecipitations, immunofluorescence and time-lapse microscopy, mutant analysis, permeabilized cell assays, proteomics, affinity chromatography, and yeast-two hybrid screens.
(1) Structure/function analysis of Rab-FIP protein complexes
Understanding the structure of proteins is the key step in determining their function in the cell. Thus, determining the properties of Rab and FIP interactions has been one of the major focuses in the lab. The current work concentrates on determining the structure of Rab-FIP complex (collaboration with Dr. Bill Weis, Stanford University) as well as kinetic properties of Rab-FIP complex formation.
Structural information from above studies is then used to analyse the role of Rab and FIP interactions in vivo. Several microscopy assays are used for that purpose. That includes fluorescent energy transfer (FRET) as well as time-lapse microscopy analysis (for cool movie depicting the fusion of transport vesicle/tubule containing GFP-labeled FIP see Figure 1).