Early events in glycosylphosphatidylinositol anchor addition. substrate proteins associate with the transamidase subunit gpi8p.
The addition of glycosylphosphatidylinositol (GPI) anchors to proteins occurs by a transamidase-catalyzed reaction mechanism soon after completion of polypeptide synthesis and translocation. We show that placental alkaline phosphatase becomes efficiently GPI-anchored when translated in the presence of semipermeabilized K562 cells but is not GPI-anchored in cell lines defective in the transamidase subunit hGpi8p. By studying the synthesis of placental alkaline phosphatase, we demonstrate that folding of the protein is not influenced by the addition of a GPI anchor and conversely that GPI anchor addition does not require protein folding. These results demonstrate that folding of the ectodomain and GPI addition are two distinct processes and can be mutually exclusive. When GPI addition is prevented, either by synthesis of the protein in the presence of cell lines defective in GPI addition or by mutation of the GPI carboxyl-terminal signal sequence cleavage site, the substrate forms a prolonged association with the transamidase subunit hGpi8p. The ability of the transamidase to recognize and associate with GPI anchor signal sequences provides an explanation for the retention of GPI-anchored protein within the ER in the absence of GPI anchor addition.
University of Manchester, School of Biological Sciences, 2.205 Stopford Building, Manchester M13 9PT, United Kingdom.
T D
TD
Spurway
J A
JA
Dalley
S
S
High
N J
NJ
Bulleid
D000217:Acyltransferases / Q000378:metabolism; D000469:Alkaline Phosphatase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D002352:Carrier Proteins / Q000378:metabolism; D015815:Cell Adhesion Molecules / Q000378:metabolism*; D005260:Female; D017261:Glycosylphosphatidylinositols / Q000378:metabolism*; D006801:Humans; D020014:K562 Cells; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010920:Placenta / Q000201:enzymology; D011247:Pregnancy; D014176:Protein Biosynthesis; D017510:Protein Folding; D021122:Protein Subunits; D011817:Rabbits; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D012156:Reticulocytes / Q000378:metabolism
D002352:Carrier Proteins; D015815:Cell Adhesion Molecules; D017261:Glycosylphosphatidylinositols; C491190:PIGK protein, human; D021122:Protein Subunits; D011994:Recombinant Proteins; D000217:Acyltransferases; C081007:COOH-terminal signal transamidase; D000469:Alkaline Phosphatase