The targeting of proteins across or into the endoplasmic reticulum (ER) is the first step in secretion and in the assembly of many membranes. With the exception of SRP, docking protein (SRP receptor) and the signal peptidase, little is known about the components involved or the requirements of this process. This is due, in large part, to the difficulty of further dissecting and reconstituting such a complex process. Augmenting a biochemical approach with a genetic one would allow further, and potentially more rapid, progress to be made in this area. Yeast cells, whose secretory pathway closely resembles that of higher eukaryotes, have the obvious advantage that they can be easily manipulated genetically. Recently targeting to the ER has been accomplished in a cell-free system derived from yeast. The objective of the research described in this proposal is the exploitation of yeast genetics, in combination with our existing biochemical expertise, to identify and characterize the components that mediate targeting to, and the translocation across, the membrane of the ER. We propose to refine the homologous yeast cell-free system to enable the isolation of cytosolic and membrane proteins involved in translocation. Important cytosolic components will be identified in a lysate-dependent post-translational assay. Biochemical and immunological methods will be employed to analyze the role of rough ER-specific proteins in translocation. Verification of the participation of these proteins in the secretory process will be accomplished in vivo by gene disruption techniques that are feasible in yeast. In parallel, we will select for new secretory mutants, defective in targeting and translocation, by the expression of a crucial cytoplasmic enzyme as a signal sequence-bearing chimera. Mutants produced in this way will then be characterized biochemically in the in vitro system.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM038538-03
Application #
3295021
Study Section
Cellular Biology and Physiology Subcommittee 1 (CBY)
Project Start
1987-09-01
Project End
1992-08-31
Budget Start
1989-09-01
Budget End
1990-08-31
Support Year
3
Fiscal Year
1989
Total Cost
Indirect Cost
Name
University of California Los Angeles
Department
Type
Schools of Medicine
DUNS #
119132785
City
Los Angeles
State
CA
Country
United States
Zip Code
90095
Benyamini, Payam; Webster, Paul; Meyer, David I (2009) Knockdown of p180 eliminates the terminal differentiation of a secretory cell line. Mol Biol Cell 20:732-44
Hyde, Maureen; Block-Alper, Laura; Felix, Jahaira et al. (2002) Induction of secretory pathway components in yeast is associated with increased stability of their mRNA. J Cell Biol 156:993-1001
Becker, F; Block-Alper, L; Nakamura, G et al. (1999) Expression of the 180-kD ribosome receptor induces membrane proliferation and increased secretory activity in yeast. J Cell Biol 146:273-84
Wanker, E E; Sun, Y; Savitz, A J et al. (1995) Functional characterization of the 180-kD ribosome receptor in vivo. J Cell Biol 130:29-39
Mayinger, P; Bankaitis, V A; Meyer, D I (1995) Sac1p mediates the adenosine triphosphate transport into yeast endoplasmic reticulum that is required for protein translocation. J Cell Biol 131:1377-86
Savitz, A J; Meyer, D I (1993) 180-kD ribosome receptor is essential for both ribosome binding and protein translocation. J Cell Biol 120:853-63
Bush, G L; Tassin, A M; Friden, H et al. (1991) Secretion in yeast. Purification and in vitro translocation of chemical amounts of prepro-alpha-factor. J Biol Chem 266:13811-4
Sanderson, C M; Meyer, D I (1991) Purification and functional characterization of membranes derived from the rough endoplasmic reticulum of Saccharomyces cerevisiae. J Biol Chem 266:13423-30
Sanderson, C M; Crowe, J S; Meyer, D I (1990) Protein retention in yeast rough endoplasmic reticulum: expression and assembly of human ribophorin I. J Cell Biol 111:2861-70
Sanz, P; Meyer, D I (1989) Secretion in yeast: preprotein binding to a membrane receptor and ATP-dependent translocation are sequential and separable events in vitro. J Cell Biol 108:2101-6

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