Characterization of a component of the yeast secretion machinery: identification of the SEC18 gene product.
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.
Eakle
K A
KA
Division of Biology, California Institute of Technology, Pasadena 91125.
Bernstein
M
M
Emr
S D
SD
D000595:Amino Acid Sequence; D001483:Base Sequence; D001692:Biological Transport; D015152:Blotting, Northern; D003001:Cloning, Molecular; D003593:Cytoplasm / Q000378:metabolism; D005656:Fungal Proteins / Q000235:genetics / Q000378:metabolism*; D005800:Genes, Fungal; D007425:Intracellular Membranes / Q000378:metabolism; D008562:Membrane Glycoproteins / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009940:Organoids / Q000378:metabolism; D012333:RNA, Messenger / Q000235:genetics; D020813:Receptor, trkB; D015183:Restriction Mapping; D012441:Saccharomyces cerevisiae / Q000235:genetics*
D005656:Fungal Proteins; D008562:Membrane Glycoproteins; D012333:RNA, Messenger; D020813:Receptor, trkB