The yeast acid phosphatase can enter the secretory pathway without its N-terminal signal sequence.
The repressible Saccharomyces cerevisiae acid phosphatase (APase) coded by the PHO5 gene is a cell wall glycoprotein that follows the yeast secretory pathway. We used in vitro mutagenesis to construct a deletion (delta SP) including the entire signal sequence and four amino acids of the mature sequence of APase. An APase-deficient yeast strain was transformed with a high-copy-number plasmid carrying the PHO5/delta SP gene. When expressed in vivo, the PHO5/delta SP gene product accumulated predominantly as an inactive, unglycosylated form located inside the cell. A large part of this unglycosylated precursor underwent proteolytic degradation, but up to 30% of it was translocated, core glycosylated, and matured by the addition of mannose residues, before reaching the cell wall. It appears, therefore, that the signal sequence is important for efficient translocation and core glycosylation of yeast APase but that it is not absolutely necessary for entry of the protein into the yeast secretory pathway. mRNA obtained by in vitro transcription of PHO5 and PHO5/delta SP genes were translated in vitro in the presence of either reticulocyte lysate and dog pancreatic microsomes or yeast lysate and yeast microsomes. The PHO5 gene product was translocated and core glycosylated in the heterologous system and less efficiently in the homologous system. We were not able to detect any translocation or glycosylation of PHO5/delta SP gene product in the heterologous system, but a very small amount of core suppression of glycosylated material could be evidenced in the homologous system.
Silve
S
S
Laboratoire de Biochimie des Porphyrines, Institut Jacques Monod, Université Paris VII, France.
Monod
M
M
Hinnen
A
A
Haguenauer-Tsapis
R
R
D000135:Acid Phosphatase / Q000235:genetics / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D001692:Biological Transport; D002872:Chromosome Deletion; D004285:Dogs; D006023:Glycoproteins / Q000378:metabolism*; D007158:Immunologic Techniques; D066298:In Vitro Techniques; D007700:Kinetics; D008861:Microsomes / Q000378:metabolism; D008970:Molecular Weight; D011499:Protein Processing, Post-Translational; D021382:Protein Sorting Signals / Q000502:physiology*; D012441:Saccharomyces cerevisiae / Q000201:enzymology*; D013329:Structure-Activity Relationship
D006023:Glycoproteins; D021382:Protein Sorting Signals; D000135:Acid Phosphatase