Proteins present within segments of the Golgi apparatus, a major subcellular organelle system within eukaryotic cells, can be considered to be either resident proteins, i.e., proteins most of whose lifetimes are spent in the organelle, or transient proteins, i.e., proteins which are processed in the organelle but whose ultimate "citizenship" is elsewhere within the cell or extracellular milieu. Based on previous results, three hypotheses can be advanced to explain residency: (1) limited entry into transport vesicles, due to restricted mobility of resident versus transient proteins, which results in the preferential inclusion of transient protein in non-selective, bulk transport vesicles budding from the Golgi apparatus; (2) preferential trapping of transient versus resident proteins in Golgi transport vesicles with the mobility of both protein classes being similar; or (3) equal inclusion of both transient and resident proteins in Golgi transport vesicles with the resident proteins then being retrieved after each round of transport. Various combinations of these hypotheses are, of course, possible. To test the validity of these hypotheses, the mobility of two membrane proteins, a transient Golgi protein, the vesicular stomatitis virus G protein, and a resident Golgi protein, a murine galactosyl transferase, will be measured directly by confocal fluorescent recovery after photobleaching (cFRAP). Recombinant DNA techniques will be used to express the specific galactosyl transferase in cultured monkey cells. The cFRAP experiments will be done in collaboration with Dr. Thomas Kreis, at the European Molecular Biology Laboratory in Heidelberg, Germany, using EMBL facilities. Preparative work for these experiments will be done at Virginia Polytechnic Institute and State University in Blacksburg, VA. In a second set of experiments, the relative enrichment of the transient and resident Golgi proteins in isolated Golgi vesicles relative to isolated Golgi complexes will be assessed. These experiments will be done entirely at VPI-SU. These experiments focus on an important problem in cell biology, namely, how do specific proteins get to their functionally appropriate destinations within the compartmentalized cell? The results of these innovative experiments are expected to advance our understanding of these critical cellular processes.
