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Characterization of a component of the yeast secretion machinery: identification of the SEC18 gene product

Eakle, Kurt A. and Bernstein, Mitchell and Emr, S.D. (1988) Characterization of a component of the yeast secretion machinery: identification of the SEC18 gene product. Molecular and Cellular Biology, 8 (10). pp. 4098-4109. ISSN 0270-7306. http://resolver.caltech.edu/CaltechAUTHORS:EAKmcb88

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Abstract

SEC18 gene function is required for secretory protein transport between the endoplasmic reticulum (ER) and the Golgi complex. We cloned the SEC18 gene by complementation of the sec18-1 mutation. Gene disruption has shown that SEC18 is essential for yeast cell growth. Sequence analysis of the gene revealed a 2,271-base-pair open reading frame which could code for a protein of 83.9 kilodaltons. The predicted protein sequence showed no significant similarity to other known protein sequences. In vitro transcription and translation of SEC18 led to the synthesis of two proteins of approximately 84 and 82 kilodaltons. Antisera raised against a Sec18-beta-galactosidase fusion protein also detected two proteins (collectively referred to as Sec18p) in extracts of 35S-labeled yeast cells identical in size to those seen by in vitro translation. Mapping of the 5' end of the SEC18 mRNA revealed only one major start site for transcription, which indicates that the multiple forms of Sec18p do not arise from mRNAs with different 5' ends. Results of pulse-chase experiments indicated that the two forms of Sec18p are not the result of posttranslational processing. We suggest that translation initiating at different in-frame AUG start codons is likely to account for the presence of two forms of Sec18p. Hydrophobicity analysis indicated that the proteins were hydrophilic in nature and lacked any region that would be predicted to serve as a signal sequence or transmembrane anchor. Although potential sites for N-linked glycosylation were present in the Sec18p sequence, the sizes of the in vivo SEC18 gene products were unaffected by the drug tunicamycin, indicating that Sec18p does not enter the secretory pathway. These results suggest that Sec18p resides in the cell cytoplasm. While preliminary cell fractionation studies showed that Sec18p is not associated with the ER or Golgi complex, association with a 100,000 x g pellet fraction was observed. This suggests that Sec18p may bind transiently to small vesicles such as those presumed to participate in secretory protein transport between ER and the Golgi complex.


Item Type:Article
Additional Information:Copyright © 1988 by the American Society for Microbiology. Received 20 May 1988/Accepted 27 June 1988 We thank Elizabeth Greyhack for help with in vitro translation, Lois Banta and Jean Edens for electron microscopy, and Michael Clark for expertise and help with microscopy in general. We also thank Shawn Westway for assistance with BIONET and Cathy Elkins for typing the manuscript. We thank Randy Schekman for support, especially in the early stages of cloning SEC18, and for continued interest throughout. This study was supported by Public Health Service grant GM-32703 from the National Institutes of Health (to S.D.E.). K.A.E. was supported in part by National Research Service Award 1 T32 GM07616 from the National Institute of General Medical Sciences. S.D.E. is a Presidential Young Investigator supported by grant DCB-8451633 from the National Science Foundation.
Record Number:CaltechAUTHORS:EAKmcb88
Persistent URL:http://resolver.caltech.edu/CaltechAUTHORS:EAKmcb88
Alternative URL:http://mcb.asm.org/cgi/content/abstract/8/10/4098
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ID Code:2405
Collection:CaltechAUTHORS
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Deposited On:03 Apr 2006
Last Modified:26 Dec 2012 08:48

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