Abstract/Details

Reconstitution of intercompartmental protein transport in yeast extracts

Baker, David.  University of California, Berkeley. ProQuest Dissertations Publishing, 1989. 9028725.

Abstract (summary)

Transport of $\alpha$-factor precursor from the endoplasmic reticulum to the Golgi complex was reconstituted in yeast extracts. Transport is measured through the coupled addition of outer-chain carbohydrate to ($\sp{35}$S)methionine-labeled $\alpha$-factor precursor translocated into the endoplasmic reticulum during a low temperature incubation. The reaction is absolutely dependent on ATP, stimulated 10-fold by cytosol, and occurs between physically separable sealed compartments. Sec mutations which block transport in vivo also block transport in vitro. Complementation of sec mutations in vitro provides a functional assay for the purification of individual intercompartmental transport factors. The 21kd GTP binding Ypt1 protein (Ypt1p) is required for ER to Golgi transport in vitro. Fractions prepared from ypt1-1 mutant cells are defective in transport and anti Ypt1 Fab fragments inhibit the wild type transport reaction. Furthermore, immunodepletion of Ypt1p from wild type fractions blocks transport. Supplementation with a large Ypt1p containing particle restores transport to immunodepleted reactions. Although the in vitro transport reaction requires physiological concentrations of calcium, Ypt1p functions independently of calcium in transport. First, buffering the free calcium at concentrations ranging from 10$\sp{-9}$ to 10$\sp{-5}$M does not relieve inhibition by Ypt1 antibodies. Second, consumption of an intermediate that accumulates in calcium deficient incubations is not inhibited by anti-Ypt1 antibodies.

Indexing (details)


Subject
Molecular biology
Classification
0307: Molecular biology
Identifier / keyword
Biological sciences
Title
Reconstitution of intercompartmental protein transport in yeast extracts
Author
Baker, David
Number of pages
140
Degree date
1989
School code
0028
Source
DAI-B 51/05, Dissertation Abstracts International
Place of publication
Ann Arbor
Country of publication
United States
University/institution
University of California, Berkeley
University location
United States -- California
Degree
Ph.D.
Source type
Dissertation or Thesis
Language
English
Document type
Dissertation/Thesis
Dissertation/thesis number
9028725
ProQuest document ID
303670112
Copyright
Database copyright ProQuest LLC; ProQuest does not claim copyright in the individual underlying works.
Document URL
https://www.proquest.com/docview/303670112