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Characterization of the SEC18 gene of S. cerevisiae : identification of a protein involved in yeast secretion

Citation

Eakle, Kurt Andrew (1989) Characterization of the SEC18 gene of S. cerevisiae : identification of a protein involved in yeast secretion. Dissertation (Ph.D.), California Institute of Technology. http://resolver.caltech.edu/CaltechETD:etd-05302007-153631

Abstract

SEC18 gene function is required for secretory protein transport between the endoplasmic reticulum and the Golgi complex. We have cloned the SEC 18 gene by complementation of the secl8-1 mutation. Deletion/disruption of this gene has shown that SEC18 is essential for yeast cell growth. Sequence analysis of the gene revealed a 2271 by open reading frame which would code for a protein of 83.9 kd. The predicted protein sequence showed no significant homology to other known protein sequences. In vitro transcription and translation of SEC18 led to the synthesis of two proteins of approximately 84 and 82 kd. Antisera raised against a Sec18-?-galactosidase fusion protein, detects two proteins from in vivo 35S labeled yeast cells identical in size to those seen by in vitro translation. Although potential sites for N-linked glycosylation are present in the Sec 18p sequence, the sizes of the in vivo SEC18 gene products are unaffected by the drug tunicamycin. Hydrophobicity analysis indicated that the protein is hydrophilic in nature and lacks any region that would be predicted to serve as a signal sequence or transmembrane anchor. These results suggest that the Secl8p resides in the cell cytoplasm. Pulse-chase experiments indicate that the two forms of Sec 18 protein are not the result of post-translational processing. Mapping of the 5' end of the SEC18 mRNA revealed only one major start site for transcription, which indicates that the multiple forms of Sec 18 protein do not arise from mRNAs with different 5' ends. We suggest that translation initiating at different in-frame AUG start codons is likely to account for the presence of two forms of the Sec 18 protein. While cell fractionation studies show that the Sec 18p are not associated with ER or Golgi compartments, association with a 100,000 x g pellet fraction has been observed suggesting that Sec 18p may bind transiently to small vesicles such as those presumed to participate in ER to Golgi transport.

Item Type:Thesis (Dissertation (Ph.D.))
Degree Grantor:California Institute of Technology
Division:Biology
Major Option:Biology
Thesis Availability:Restricted to Caltech community only
Research Advisor(s):
  • Unknown, Unknown
Thesis Committee:
  • Unknown, Unknown
Defense Date:1 October 1988
Record Number:CaltechETD:etd-05302007-153631
Persistent URL:http://resolver.caltech.edu/CaltechETD:etd-05302007-153631
Default Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:2312
Collection:CaltechTHESIS
Deposited By: Imported from ETD-db
Deposited On:31 May 2007
Last Modified:26 Dec 2012 02:49

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