Proteins that are destined for export (secretion) from the cell and proteins that are destined to become integral components of either the plasma membrane or certain intracellular membranes (Golgi apparatus, lysosomes) undergo special routing and proteolytic processing within the cell. These routing and processing steps are critical to the proper function and ultimate location of the proteins. These proteins are originally synthesized in the cytoplasm bearing "signal peptides" at their amino termini. The "signal peptide" serves as the targeting sequence for directing the nascent protein to an intracellular membrane-bounded compartment termed the endoplasmic reticulum (ER). This "signal peptide" directs the translocation of the protein through the ER membrane, from the cytoplasm into the lumen of the ER. Once in the lumen, this "signal peptide" is proteolytically removed from the rest of the protein. Subsequent maturation and movement of the protein to its final location proceeds from there. Thus, protein translocation across the ER membrane and signal peptide cleavage are important early steps in the biogenesis of many essential proteins. Numerous maturation steps occur in the ER within seconds after translocation, suggesting that the enzymes and protein complexes mediating these steps are probably present at or near the translocation site, which includes the protein-conducting channel formed by the trimeric Sec61 complex, the signal peptidase complex (SPC), and Kar2p (yeast BiP), a molecular chaperone that appears to function in both translocation and maturation. It is likely that interactions or coordination between these enzymes and protein complexes is occurs, since protein maturation events may need to be coordinated with each other and with the translocation process to ensure that maturation events occur in the proper sequential steps. Recently, Dr. Fang observed an interaction between the SPC and Kar2p using genetic and biochemical approaches. The presence of this interaction leads to the questions, do other proteins involved in translocation and maturation interact, and what is the functional significance of this and possibly other interactions? To address these questions, the following scientific aims will be pursued, using in vivo analysis of the yeast Saccharomyces cerevisiae: Aim 1, to conduct structure - function analysis of the SPC and Kar2p interaction by identifying the interacting subunits or domains and to determine the importance of this interaction for signal peptide processing and translocation; Aim 2, to determine whether the SPC and Sec61 complex interact in yeast and to discern whether an interaction between these proteins is important for signal peptide processing and translocation; and Aim 3, to isolate and identify new proteins that interact with the SPC using a broad-based proteomic approach.
This Faculty Early Career Development Award (CAREER) also includes a component that integrates research with education. This part of the project will involve the teaching and training of graduate students in the areas of intracellular trafficking and proteomics at two institutions in Nashville, Tennessee: Dr. Fang's home institution, Vanderbilt University, and Meharry Medical College, a historically black institution. The educational objectives of this project are: 1, to modernize an existing Advanced Microbial Genetics course at Vanderbilt that is also attended by students from Meharry by integrating new material to reflect the impact and rapid progress of the microbial genome project and related new cutting-edge technologies; 2, to outreach under-represented groups by lecturing to graduate students in the Meharry Membrane Biochemistry course as an adjunct assistant professor at that institution and by hosting students from Meharry as laboratory trainees during the summers; and 3, to mentor graduate students as well as train undergraduate and high school students by providing them with hands-on research experiences in the laboratory.
