INTRACELLULAR TRANSPORT OF GLYCOPROTEINS

We propose to pursue three related projects aimed at increasing our understanding of the mechanisms involved in the transport and sorting of proteins made on the rough endoplasmic reticulum. 1) Compartmentalization of the endoplasmic reticulum: The smooth endoplasmic reticulum (SER) lacks the ribosomes and their associated membrane proteins and has a totally different structure. We have demonstrated that the G protein of VSV has free access to and egress from the SER of UT-1 cells. We plan to use electon microscopy an autoradiography test whether transport from the RER to the SER is unidirectional, to measure its rate, and to examine its energy requirements. 2) Mechanism(s) involved in transport of proteins made on the RER to lysosomes: It is now known that a specific phosphomannosyl recognition marker is added to many lysosoma proteins and is responsible for their transport to lysosomes. It is not known, however, how cells decide which proteins should receive this marker. We will use cloned cDNA expression vectors to express normal and altered forms of lysosomal enzyme beta-glucuronidase to ask: What is the primary signal responsible for the addition of the phosphomannosyl recognition marker? Is one signal responsible for the addition of the recognition marker to all asparagine-linked oligosaccharides on a protein? What is the effect of membrane association of a lysosmal protein on its transport to lysosomes? 3) Transport between the RER and compartments of the Golgi apparatus: We have characterized an altered form of the G protein (del-1554) that is transported from the RER to the Golgi apparatus, but fails to reach the cell surface. The proteins probably accumulate at the medial region of the Golgi apparatus. The defect in del-1554 has been localized to 12 amino acids at its carboxy terminus. Our data suggest that del-1554 may interact with host cell component(s) involved in intracellular protein transport. We will undertake experiments to localize the exact site at which these molecules accumulate. We will test whether del-1554 inhibits the intracellular transport of other membrane and secreted proteins, and whether other membrane proteins modified to contain the 12 amino acid "tail" accumulate at the same intracellular sites. If the "tail" has these activities, we plan to use an oligopeptide with this structure to isolate the host cell component(s) involved.