The objective of this research proposal is to identify and characterize components of the ER sorting machinery involved in COPII-directed cargo selection and concentration. ER pre-budding complexes enriched in low molecular weight proteins which may belong to the p24 family have been identified. p24 proteins may select and concentrate cargo prior to ER export. This proposal will test the hypotheses that low molecular weight proteins are members of the p24 family and that p24 proteins play a key role in COP II-directed cargo selection and concentration.
Specific aim #1 : Determine the identity of low molecular weight ER pre-budding complex proteins. Purified proteins will be subjected to Edman degradation and degenerate oligonucleotides will be used to isolate full length cDNA's by PCR and library screening.
Specific aim #2 : Investigate the role of low molecular weight proteins and known p24 proteins in COPII-directed cargo selection and concentration. Reagents directed toward the cytoplasmic and lumenal domains of these proteins will be used to examine their role in COPII-directed cargo selection/concentration in vitro by quantitating a) incorporation of VSV-G cargo into COPII-coated vesicles and b) VSV-G enrichment in ER pre-budding complexes, and in vivo by quantitating a) VSV-G trafficking from the ER to the Golgi apparatus and b) VSV-G green fluorescent protein concentration in ER exit foci in living cells by real-time fluorescence microscopy. It is anticipated that data from this proposal will ultimately help understand and treat human diseases involving defective protein trafficking from the ER.
