title: Yeast carboxypeptidase Y requires glycosylation for efficient intracellular transport, but not for vacuolar sorting, in vivo stability, or activity.
abstract: Functions of the carbohydrate side chains of the yeast vacuolar enzyme carboxypeptidase Y (CPY) were investigated by removal, through site-directed mutagenesis, of the sequences which act as signals for N-linked glycosylation. The mutant forms of the enzyme were analysed with respect to activity and intracellular sorting, and the stabilities in vivo and in vitro were studied. It was found that carbohydrate was not important for accurate vacuolar targeting of CPY, but that the rate of transport of the unglycosylated CPY through the secretory pathway to the vacuole was reduced. Tunicamycin, which inhibits the formation of asparagine-linked glycosylation, had a similar effect on the transport of CPY at 23 degrees C. However, the absence of N-linked carbohydrate in general had the more dramatic result of blocking the transport of CPY altogether at an increased temperature (37 degrees C). The unglycosylated mutant CPY was not temperature sensitive for transport in the absence of tunicamycin. Analysis of mutant enzymes containing a single glycosyl residue at each of the four positions showed that the residue at position 87 was particularly important for transport. There was no decrease in the intracellular stability of the completely unglycosylated enzyme, and in vitro the rate of heat inactivation of this species was not increased.
authors.affiliation: Department of Yeast Genetics, Carlsberg Laboratory, Copenhagen Valby, Denmark.
authors.forename: J R
authors.initials: JR
authors.lastname: Winther
authors.forename: T H
authors.initials: TH
authors.lastname: Stevens
authors.forename: M C
authors.initials: MC
authors.lastname: Kielland-Brandt
mesh_terms: D001483:Base Sequence; D001692:Biological Transport; D002268:Carboxypeptidases / Q000378:metabolism*; D004795:Enzyme Stability; D006031:Glycosylation; D008969:Molecular Sequence Data; D009154:Mutation; D014415:Tunicamycin / Q000494:pharmacology; D014617:Vacuoles / Q000201:enzymology*; D015003:Yeasts / Q000201:enzymology*
chemical_list: D014415:Tunicamycin; D002268:Carboxypeptidases; C022633:serine carboxypeptidase