Expression of mouse cathepsin L cDNA in Saccharomyces cerevisiae

Evidence that cathepsin L is sorted for targeting to yeast vacuole

Yukio Nishimura, Keitaro Kato

Research output: Contribution to journalArticle

11 Citations (Scopus)

Abstract

To investigate the intracellular transport mechanism of lysosomal cathepsin L in yeast cells, we attempted to produce mouse cathepsin L in Saccharomyces cerevisiae by placing the coding region under the control of the promoter of the yeast glyceraldehyde 3-phosphate dehydrogenase (GAPDH) gene. Immunoblotting analysis by the use of an antibody specific for rat cathepsin L revealed that the yeast cells carrying the cathepsin L coding sequence produced 39- and 30-kDa products, which correspond to the rat procathepsin L and the single-chain form of mature cathepsin L, respectively. The precursor polypeptide showed sensitivity toward endoglycosidase H treatment. Cell fractionation experiments demonstrated that the processed form of 30-kDa cathepsin L was found to be colocalized to the yeast vacuole with the marker enzyme carboxypeptidase Y in a Ficoll step gradient. In the prepared vacuolar fraction, a considerable amount of cathepsin L was revealed to be cofractionated with the vacuolar membranes. Furthermore, the phase separation experiments with Triton X-114 provide the first evidence showing that the mature form of cathepsin L polypeptide is strongly associated with the vacuolar membranes. Therefore, the present results suggest that the mouse cathepsin L precursor polypeptide is initially synthesized as the proenzyme in the yeast cells and then correctly delivered to the vacuole. During the intracellular sorting pathway, the procathepsin L would undergo the post-translational proteolytic processing step to generate the mature enzyme. Based on these lines of evidence, we propose that cathepsin L is recognized by mechanisms similar to those for the intracellular sorting and processing of vacuolar proteins in the yeast cells.

Original languageEnglish
Pages (from-to)318-324
Number of pages7
JournalArchives of Biochemistry and Biophysics
Volume298
Issue number2
DOIs
Publication statusPublished - Nov 1 1992

Fingerprint

Cathepsin L
Vacuoles
Yeast
Saccharomyces cerevisiae
Complementary DNA
Yeasts
Cells
Sorting
Peptides
Cathepsin A
Membranes
Cell Fractionation
Ficoll
Enzyme Precursors
Glyceraldehyde-3-Phosphate Dehydrogenases
Fungal Proteins
Glycoside Hydrolases
Enzymes
Fractionation
Processing

All Science Journal Classification (ASJC) codes

  • Biophysics
  • Biochemistry
  • Molecular Biology

Cite this

Expression of mouse cathepsin L cDNA in Saccharomyces cerevisiae : Evidence that cathepsin L is sorted for targeting to yeast vacuole. / Nishimura, Yukio; Kato, Keitaro.

In: Archives of Biochemistry and Biophysics, Vol. 298, No. 2, 01.11.1992, p. 318-324.

Research output: Contribution to journalArticle

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