): Biogenesis of a membrane protein is a dynamic process with multiple steps occurring in parallel. Defects in membrane protein biosynthesis are manifested in several genetic diseases, and may be pivotal to understanding many more. However, before lesions of membrane protein biogenesis can be discovered, mechanisms which describe the fate of a membrane protein must first be found. Using the multipass protein opsin, experiments are proposed which separate translation, membrane targeting, translocation, and integration. The ability to separate the various steps of opsin biogenesis hinges on the observation that mRNAs that lack stop codons are translated by the ribosome but are not released. In the case of translation of truncated mRNAs for opsin, the arrested ribosomes with their nascent polypeptides bound are still capable of interacting with the translocation machinery in vitro. These truncated proteins can undergo targeting, translocation, and integration as assayed by signal recognition peptide (SRP) binding and glycosylation. Using the unique biochemical properties of arrested ribosomes three experimental aims will be addressed. First, when does the signal recognition particle interact with the first transmembrane domain of opsin? Second, what is the minimum amount of sequence required to support targeting and translocation of the nascent chain? Third, when during translation is opsin competent to translocate across the ER? It is hoped that answers to these questions will be more generally applicable to the biosynthesis of other multiple pass proteins.

Agency
National Institute of Health (NIH)
Institute
National Eye Institute (NEI)
Type
Postdoctoral Individual National Research Service Award (F32)
Project #
5F32EY007081-03
Application #
6623222
Study Section
Visual Sciences C Study Section (VISC)
Program Officer
Mariani, Andrew P
Project Start
2001-11-01
Project End
Budget Start
2002-11-01
Budget End
2003-10-31
Support Year
3
Fiscal Year
2003
Total Cost
$48,148
Indirect Cost
Name
Rockefeller University
Department
Biology
Type
Other Domestic Higher Education
DUNS #
071037113
City
New York
State
NY
Country
United States
Zip Code
10065