A defect in glycosylphosphatidylinositol (GPI) transamidase activity in mutant K cells is responsible for their inability to display GPI surface proteins.
The final step in the pathway that provides for glycosylphosphatidylinositol (GPI) anchoring of cell-surface proteins occurs in the lumen of the endoplasmic reticulum and consists of a transamidation reaction in which fully assembled GPI anchor donors are substituted for specific COOH-terminal signal peptide sequences contained in nascent polypeptides. In previous studies we described a human K562 cell mutant line, designated class K, which assembles all the known intermediates of the GPI pathway but fails to display GPI-anchored proteins on its surface membrane. In the present study, we used mRNA encoding miniPLAP, a truncated form of placental alkaline phosphatase (PLAP), in in vitro assays with rough microsomal membranes (RM) of mutant K cells to further characterize the biosynthetic defect in this line. We found that RM from mutant K cells supported NH2-terminal processing of the nascent translational product, preprominiPLAP, but failed to show any detectable COOH-terminal processing of the resulting prominiPLAP to GPI-anchored miniPLAP. Proteinase K protection assays verified that NH2-terminal processed prominiPLAP was appropriately translocated into the endoplasmic reticulum lumen. The addition of hydrazine or hydroxylamine, which can substitute for GPI donors, to RM from wild-type or mutant cells defective in various intermediate biosynthetic steps in the GPI pathway produced large amounts of the hydrazide or hydroxamate of miniPLAP. In contrast, the addition of these nucleophiles to RM of class K cells yielded neither of these products. These data, taken together, lead us to conclude that mutant K cells are defective in part of the GPI transamidase machinery.
Chen
R
R
Institute of Pathology, Case Western Reserve University, Cleveland, OH 44106, USA.
Udenfriend
S
S
Prince
G M
GM
Maxwell
S E
SE
Ramalingam
S
S
Gerber
L D
LD
Knez
J
J
Medof
M E
ME
D000217:Acyltransferases / Q000378:metabolism; D000469:Alkaline Phosphatase; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D058851:GPI-Linked Proteins; D017261:Glycosylphosphatidylinositols / Q000378:metabolism*; D006801:Humans; D006834:Hydrazines / Q000494:pharmacology; D007527:Isoenzymes / Q000378:metabolism; D008970:Molecular Weight; D009154:Mutation; D011499:Protein Processing, Post-Translational / Q000187:drug effects
D058851:GPI-Linked Proteins; D017261:Glycosylphosphatidylinositols; D006834:Hydrazines; D007527:Isoenzymes; C029424:hydrazine; D000217:Acyltransferases; C081007:COOH-terminal signal transamidase; D000469:Alkaline Phosphatase; C019839:alkaline phosphatase, placental