Receptor-mediated endocytosis plays an Important role in allowing mammalian cells to sense and respond to their environment. During the last ten years, significant progress has been made in understanding this fundamental process, but a number of questions remain unanswered. Continuation of studies of the endocytic system is proposed. These studies will combine fluorescence spectroscopy of living cells, somatic cell genetics and cell-free analysis of endocytic compartments. A new class of endocytosis mutants, those with defects in recycling from endosomes to the plasma membrane, will be isolated by fluorescence-activated cell sorting. Initial members of this class of mutants have been isolated, demonstrating the feasibility of this approach. The characteristics of these mutants will be determined, and the genes responsible for the defect(s) will be isolated. The long term goals of this aspect of the project are to understand the processes of receptor recycling and endosomal fission events at the molecular level. Previous work on this project has demonstrated that the Na,K-ATPase plays a role in regulating endosomal pH in living cells. This phenomenon occurs in some, but not all, cell types. A second goal of this proposal is understanding the means by which the traffic of Na,K-ATPase to (and possibly from) endosomes occurs in various cell types.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM032508-08
Application #
2176613
Study Section
Molecular Cytology Study Section (CTY)
Project Start
1983-08-01
Project End
1994-08-31
Budget Start
1992-09-01
Budget End
1993-08-31
Support Year
8
Fiscal Year
1992
Total Cost
Indirect Cost
Name
Carnegie-Mellon University
Department
Biology
Type
Schools of Arts and Sciences
DUNS #
052184116
City
Pittsburgh
State
PA
Country
United States
Zip Code
15213
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Brockman, S A; Murphy, R F (1994) Isolation and analysis of somatic cell mutants with defects in endocytic traffic. Methods Cell Biol 42 Pt B:131-48
Murphy, R F; Schmid, J; Fuchs, R (1993) Endosome maturation: insights from somatic cell genetics and cell-free analysis. Biochem Soc Trans 21 ( Pt 3):716-20
Wilson, R B; Mastick, C C; Murphy, R F (1993) A Chinese hamster ovary cell line with a temperature-conditional defect in receptor recycling is pleiotropically defective in lysosome biogenesis. J Biol Chem 268:25357-63
Cain, C C; Wilson, R B; Murphy, R F (1991) Isolation by fluorescence-activated cell sorting of Chinese hamster ovary cell lines with pleiotropic, temperature-conditional defects in receptor recycling. J Biol Chem 266:11746-52
Sipe, D M; Jesurum, A; Murphy, R F (1991) Absence of Na+,K(+)-ATPase regulation of endosomal acidification in K562 erythroleukemia cells. Analysis via inhibition of transferrin recycling by low temperatures. J Biol Chem 266:3469-74
Sipe, D M; Murphy, R F (1991) Binding to cellular receptors results in increased iron release from transferrin at mildly acidic pH. J Biol Chem 266:8002-7
Roederer, M; Barry, J R; Wilson, R B et al. (1990) Endosomes can undergo an ATP-dependent density increase in the absence of dense lysosomes. Eur J Cell Biol 51:229-34
Bowser, R; Murphy, R F (1990) Kinetics of hydrolysis of endocytosed substrates by mammalian cultured cells: early introduction of lysosomal enzymes into the endocytic pathway. J Cell Physiol 143:110-7
Yamashiro, C T; Kane, P M; Wolczyk, D F et al. (1990) Role of vacuolar acidification in protein sorting and zymogen activation: a genetic analysis of the yeast vacuolar proton-translocating ATPase. Mol Cell Biol 10:3737-49

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