Characterization of the Hansenula polymorpha CPY gene encoding carboxypeptidase Y

• Published in: Yeast (Chichester, England) Vol. 15; no. 3; pp. 181 - 189
• Main Authors: Bellu, A. R., van der Klei, I. J., Rechinger, K. B., Yavuz, M., Veenhuis, M., Kiel, J. A. K. W.
• Format: Journal Article
• Language: English
• Published: Chichester, UK John Wiley & Sons, Ltd 01.02.1999
• Subjects: Amino Acid Sequence; autophagy; Blotting, Western; Carboxypeptidases - chemistry; Carboxypeptidases - genetics; Carboxypeptidases - metabolism; Cathepsin A; Cloning, Molecular; Genes, Fungal - genetics; Genomic Library; Glycoside Hydrolases - metabolism; Glycosylation; Hansenula polymorpha; Immunohistochemistry; methylotrophic yeast; Microbodies - metabolism; Microbodies - physiology; Molecular Sequence Data; Molecular Weight; Mutagenesis, Insertional; Open Reading Frames - genetics; peroxisome turnover; Pichia - cytology; Pichia - enzymology; Pichia - genetics; Pichia - growth & development; Protein Sorting Signals - genetics; Recombinant Fusion Proteins - chemistry; Recombinant Fusion Proteins - metabolism; Sequence Alignment; Sequence Deletion; Sequence Homology, Nucleic Acid; vacuolar proteinases; Vacuoles - metabolism
• ISSN: 0749-503X; 1097-0061
• DOI: 10.1002/(SICI)1097-0061(199902)15:3<181::AID-YEA355>3.0.CO;2-Y

We have isolated the Hansenula polymorpha CPY gene encoding carboxypeptidase Y (Hp‐CPY). The deduced amino acid sequence revealed that Hp‐CPY consists of 541 amino acids and has a calculated Mr of 60,793. The protein is highly similar to Saccharomyces cerevisiae CPY (61·8% identity). At the N‐terminus of Hp‐CPY signals for the entry into the secretory pathway and subsequent sorting to the vacuole were identified. Immunocytochemically, using monospecific antibodies raised against Hp‐CPY, the protein was localized to the vacuole. On Western blots, a diffuse protein band was observed in extracts of H. polymorpha cells, suggesting that the protein is glycosylated. This was confirmed by endoglycosidase H treatment, which resulted in a strong reduction of the apparent Mr of the protein. We have investigated the effect of CPY deletion on the degradation of peroxisomes, an autophagous process that occurs when the organelles become redundant for growth. In Δcpy cells peroxisomal proteins were degraded in the vacuole as efficiently as in wild‐type H. polymorpha cells, indicating that CPY is not a major proteinase in this pathway. Copyright © 1999 John Wiley & Sons, Ltd.