The pathway by which mammalian plasma membrane proteins and lipids are degraded will be investigated in hepatoma cultures. To determine the degree of synchrony by which membrane proteins are internalized and degraded, cells will be pulse labeled with isotopic amino acids, chased, pulsed with a second isotope, haptenized with trinitrobenzene sulfonate, immunoprecipitated with anti-hapten antibody, precipitated proteins separated by polyacrylamide gel electrophoresis, and the osotope ratio for each determined. The degradation of membrane lipids will be assessed in a similar manner after cells are pulse-labeled with radio-labeled palmitate, extracted, and lipid classes separated by thin layer chromatography. Another completely different approach to the same problem will be to purify in situ labeled membrane proteins (e.g., red cell band 3, glycophorin, or bacteriophage M13 coat protein), to inset the labeled protein into virosomes composed of defined lipid and either vesicular stomatitis or Sendai virus envelope proteins, and to then fuse the virosome to recipient cells by pH or polyethylene glycol treatment. The degradation rate of implanted membrane proteins will then be autoradiographically determined after electrophoretic separation of recipient cell proteins. The cellular compartment for ultimate degradation of implanted proteins will be identified by labeling proteins with (C14) sucrose prior to their implantation, and then fractionating chased cells into lysosome and cytosolic compartments. Membrane impermeant C14-sucrose amino acids will then accumulate in the compartmentalized site of ultimate degradation. To determine the extent to which pinocytic vesicle proteins are representative of those present on the cell surface, lactoperoxidase taken up into cells by fluid phase pinocytosis will be used to iodinate pinocytic vesicle proteins. Two-dimensional gels of these proteins will be compared to those produced from proteins labeled by extracellular lactoperoxidase. Lastly the effect of protein perturbants on subsequent degradation of membrane proteins will be investigated. Perturbants include membrane peroxidation (generated by addition of light, rotenone, or malondialdehyde), deglycosylation, glycosylation, and proteolytic cleavage. By these studies the pathway and limiting steps in membrane degradation will be dissected. Information gained may help to understand the metabolic stability of membrane receptors, transporters, and enzymes in both normal and diseased states.
| Hall, R E; Hare, J F (1990) Respiratory chain-linked NADH dehydrogenase. Mechanisms of assembly. J Biol Chem 265:16484-90 |
| Hare, J F (1989) Turnover and compartmentation of gp70/p15 E in psi 2 cells. Biochem Biophys Res Commun 161:596-603 |
| Hare, J F; Lee, E (1989) Metabolic behavior of cell surface biotinylated proteins. Biochemistry 28:574-80 |
| Hare, J F (1988) Dissection of membrane protein degradation mechanisms by reversible inhibitors. J Biol Chem 263:8759-64 |
| Rivett, A J; Hare, J F (1987) Enhanced degradation of oxidized glutamine synthetase in vitro and after microinjection into hepatoma cells. Arch Biochem Biophys 259:423-30 |
| Hare, J F; Huston, M (1985) Virosome-mediated implantation of red cell band 3 into the plasma membrane of cultured hepatoma cells. Exp Cell Res 161:317-30 |
