Expression of Rat Cathepsin D cDNA in Saccharomyces cerevisiae: Implications for Intracellular Targeting of Cathepsin D to Vacuoles^1

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Author(s)

    • NISHIMURA Yukio
    • Division of Physiological Chemistry, Faculty of Pharmaceutical Sciences Graduate School of Medical Sciences, Kyushu University
    • TAKESHIMA Hiroaki
    • Division of Physiological Chemistry, Faculty of Pharmaceutical Sciences Graduate School of Medical Sciences, Kyushu University
    • SAKAGUCHI Masao
    • Department of Molecular Biology, Graduate School of Medical Sciences, Kyushu University
    • MIHARA Katsuyoshi
    • Department of Molecular Biology, Graduate School of Medical Sciences, Kyushu University
    • OMURA Tsuneo
    • Department of Molecular Biology, Graduate School of Medical Sciences, Kyushu University
    • KATO Keitaro
    • Division of Physiological Chemistry, Faculty of Pharmaceutical Sciences Graduate School of Medical Sciences, Kyushu University
    • HIMENO Masaru
    • Division of Physiological Chemistry, Faculty of Pharmaceutical Sciences Graduate School of Medical Sciences, Kyushu University

Abstract

To investigate the intracellular transport mechanisms of lysosomal cathepsin D in yeast cells, we produced cathepsin D in <i>Saccharomyces cerevisiae</i> 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 D coding sequence produced an intermediate species which had a slightly higher molecular weight than that of the mature cathepsin D. Cell fractionation experiments demonstrated that the cathepsin D polypeptide was colocalized to the yeast vacuole with the marker enzyme carboxypeptidase Y in a Ficoll step gradient. A biosynthesis study with pulse-chase kinetic analysis revealed that the precursor polypeptide was accurately sorted to the yeast vacuoles as determined by cell fractionation, and that <i>N</i>-linked carbohydrate modifications were not required for vacuolar sorting of this protein. To elucidate the role of the propeptide region of cathepsin D, which might function in the intracellular targeting to the vacuole, a deletion mutant of cathepsin D lacking the propeptide was prepared and its intracellular targeting was examined after transfection into yeast cells. Immunoblotting analysis demonstrated that the propeptide-deleted mutant protein was recovered in a low quantity as compared with that in the case of yeast cells expressing the wild-type protein in the isolated vacuolar fraction. Immunofluorescence analysis revealed that the deletion mutant protein appeared to be accumulated within the intracellular small vesicles but not in the carboxypeptidase Y-positive vacuoles. Overall, these results indicate that the rat cathepsin D precursor polypeptide is recognized by mechanisms similar to those involved in the intracellular sorting of vacuolar proteins through the ER/Golgi/vacuolar sorting pathway in yeast cells, and that the propeptide has an important function in translocation of the cathepsin D polypeptide to the vacuole.

Journal

  • The Journal of Biochemistry

    The Journal of Biochemistry 118(1), 168-177, 1995-07-01

    The Japanese Biochemical Society

References:  40

Cited by:  1

Codes

  • NII Article ID (NAID)
    10005183071
  • NII NACSIS-CAT ID (NCID)
    AA00694073
  • Text Lang
    ENG
  • Article Type
    Journal Article
  • ISSN
    0021924X
  • Data Source
    CJP  CJPref  J-STAGE 
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