Yeast carboxypeptidase Y requires glycosylation for efficient intracellular transport, but not for vacuolar sorting, in vivo stability, or activity

TY - JOUR

T1 - Yeast carboxypeptidase Y requires glycosylation for efficient intracellular transport, but not for vacuolar sorting, in vivo stability, or activity

AU - Winther, Jakob R.

AU - Stevens, T H

AU - Kielland-Brandt, Morten

PY - 1991

Y1 - 1991

N2 - 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.

AB - 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.

KW - Base Sequence

KW - Biological Transport

KW - Carboxypeptidases

KW - Enzyme Stability

KW - Glycosylation

KW - Molecular Sequence Data

KW - Mutation

KW - Tunicamycin

KW - Vacuoles

KW - Yeasts

M3 - Journal article

C2 - 2029899

SN - 1742-464X

VL - 197

SP - 681

EP - 689

JO - FEBS Journal

JF - FEBS Journal

IS - 3

ER -