Characterization of SEC14: A gene essential for secretion in Saccharomyces cerevisiae
Malehorn, David Edward
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Permalink
https://hdl.handle.net/2142/20146
Description
Title
Characterization of SEC14: A gene essential for secretion in Saccharomyces cerevisiae
Author(s)
Malehorn, David Edward
Issue Date
1990
Department of Study
Microbiology
Discipline
Microbiology
Degree Granting Institution
University of Illinois at Urbana-Champaign
Degree Name
Ph.D.
Degree Level
Dissertation
Keyword(s)
Biology, Molecular
Language
eng
Abstract
SEC14, a Saccharomyces cerevisiae gene whose product is required for export of yeast secretory proteins from the Golgi complex, was cloned and characterized. SEC14 was shown to be an essential gene for vegetative growth. Nucleotide sequencing revealed SEC14 to be an intron-containing gene, which encodes a 35 kD protein. Sec14p was demonstrated to be nonglycosylated, and to behave primarily as a cytosolic protein. Structurally related proteins were detected in yeasts Schizosaccharomyces pombe and Kluyveromyces lactis. The homologous Sec14p from Kluyveromyces lactis was shown to functionally complement sec14$\sp{\rm ts}$ defects in S. cerevisiae, suggesting a degree of conservation of Sec14p structure and function.
The sites of SEC14 transcription initiation and termination were characterized. Primer extension analysis revealed multiple start sites 17 to 53 bp upstream of the SEC14 coding region. A start site 29 bp upstream was observed in the SEC14 cDNA, which was also shown to lack the intron sequence. A TATA like sequence 101 to 108 bp upstream of the coding region was suggested to control transcription initiation, and this identification was supported by the finding that deletion of this sequence is a lethal event. The 3$\sp\prime$ terminus of the SEC14 message was determined to be more than 180 bp downstream of the stop codon. Comparison of expression of varied promoter-truncated derivatives of SEC14 suggests that sufficient transcriptional regulatory sequences reside within 136 bp upstream of the SEC14 gene. In addition, yeast cells were shown to tolerate a severe reduction in Sec14p levels.
The sec14$\sp{\rm ts}$mutation was shown to be due to a G $>$ A transition near the carboxy terminus of the SEC14 coding region, converting glycine to aspartate. The mutation confers aspects of both thermolability for function as well as temperature sensitivity for synthesis upon Sec14p. Alternate temperature sensitive alleles of SEC14 were generated in vitro by introducing a variety of substitutions at the site of the original sec14 mutation.
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