Anchoring of membrane proteins via phosphatidylinositol is deficient in two classes of Thy-1 negative mutant lymphoma cells.

Abstract

Recent evidence shows that mature Thy‐1 glycoprotein lacks amino acids 113‐143 predicted from the cDNA sequence and is anchored to the plasma membrane by a phosphatidylinositol‐containing glycolipid attached to amino acid 112. Previously characterized Thy‐1‐deficient mutant lymphoma lines of complementation classes A and E were analysed. They make detergent binding Thy‐1 precursors but, in contrast to wild‐type, the detergent binding moiety cannot be removed by phospholipase C. Moreover, tryptophan which only occurs at position 124 is incorporated into mutant but not parental Thy‐1. This suggests that the mutants make a Thy‐1 precursor of 143 amino acids but fail to replace its C‐terminal end by a glycolipid anchor.

Glycolipid anchors are attached to Thy-1 glycoprotein rapidly after translation

“…We find that, by this criterion, the attachment of glycolipid anchors occurs with similar kinetics as in trypanosomes. We will discuss the implications of this finding for the understanding of the phenotype of mutant thymoma cells which fail to replace the C-terminal hydrophobic amino acids of Thy-I by a glycolipid anchor [27,28].…”

“…Detergent phases from labelled cells were re-extracted with 1 ml of 0.1 M-Tris/HCl (pH 7.4)/0.05 m-NaCl/ 0.25 M-x-methyl mannoside/ 1 mM-EDTA and proteinase inhibitors as described previously [27] and treated in the same buffer with PLC (Bacillus cereus, Type III, Sigma). In some cases, PLC was inactivated by boiling in 10 mmTris/ 150 mM-NaCl for 15 min before being added to cell lysates.…”

“…In the present study, we chose the Thy-I antigen in order to investigate the kinetics of addition of glycolipid anchors to surface glycoproteins in a mammalian system. Thy-I is a major surface glycoprotein on brain cells, thymocytes and Tlymphomas, and the presence of glycolipid anchors can be monitored using the change of the partitioning of the molecule in Triton X-1 14 (TX-1 14) solutions upon PLC treatment [26,27]. We find that, by this criterion, the attachment of glycolipid anchors occurs with similar kinetics as in trypanosomes.…”

“…were removed by centrifugation at 5000 g for 10 s, tubes were immediately placed on ice, the labelling medium was removed and cells lysed in 1 00 TX-114 as described previously [27]. Spent labelling medium was briefly sonicated to destroy remaining cells and then used for a further round of labelling.…”

“…2 below). After lysis for 30-60 min on ice, soluble and membrane proteins were separated and re-extracted as described previously [27]. In some experiments (Fig.…”