Acylation of Glucosaminyl Phosphatidylinositol Revisited

doi:10.1074/jbc.271.43.27031

Acylation of Glucosaminyl Phosphatidylinositol Revisited

PALMITOYL-CoA DEPENDENT PALMITOYLATION OF THE INOSITOL RESIDUE OF A SYNTHETIC DIOCTANOYL GLUCOSAMINYL PHOSPHATIDYLINOSITOL BY HAMSTER MEMBRANES PERMITS EFFICIENT MANNOSYLATION OF THE GLUCOSAMINE RESIDUE*

From the Departments of ^‡ Pharmacology and
^¶ Molecular Genetics and the
^§ Cell Regulation Graduate Program, The University of Texas Southwestern Medical Center, Dallas, Texas 75235-9041

^∥ To whom correspondence should be addressed. Tel.: 214-648-2323; Fax: 214-648-2971; E-mail: LEHRMAN{at}UTSW.SWMED.EDU.

Abstract

Two critical steps in the assembly of yeast and mammalian glycosylphosphatidylinositol (GPI) anchor precursors are palmitoylation of the inositol residue and mannosylation of the glucosamine residue of the glucosaminyl phosphatidylinositol (GlcNα-PI) intermediate. Palmitoylation has been reported to be acyl-CoA dependent in yeast membranes (Costello, L. C., and Orlean, P. (1992) J. Biol. Chem. 267, 8599-8603) but strictly acyl-CoA independent in rodent membranes (Stevens, V. L., and Zhang, H. (1994) J. Biol. Chem. 269, 31397-31403), and thus poorly conserved. In addition, it was suggested that acylation must precede mannosylation in both yeast (Costello, L. C., and Orlean, P. (1992) J. Biol. Chem. 276, 8599-8603) and rodent (Urakaze, M., Kamitani, T., DeGasperi, R., Sugiyama, E., Chang, H.-M., Warren, C. D., and Yeh, E. T. H. (1992) J. Biol. Chem. 267, 6459-6462) cells because GlcNα-acyl-PI accumulates in vivo when mannosylation is blocked. However, GlcNα-acyl-PI accumulation would also be expected if mannosylation and acylation were independent of each other.

These issues were addressed by the use of a synthetic dioctanoyl GlcNα-PI analogue (GlcNα-PI(C8)) as an in vitro substrate for GPI-synthesizing enzymes in Chinese hamster ovary cell membranes. GlcNα-PI(C8) was acylated in an manner requiring acyl-CoA. Thus, the process involving acyl-CoA reported for yeast has been conserved in mammals. Furthermore, both GlcNα-PI(C8) and GlcNα-acyl-PI(C8) could be mannosylated in vitro, but mannosylation of the latter was significantly more efficient. This provides direct support for the earlier suggestion that acylation precedes mannosylation in rodents cells. A similar result was also observed with the Saccharomyces cerevisiae mannosyltransferase.

In contrast, it has been reported that mannosylation of endogenous GlcNα-PI by Trypansoma brucei membranes occurs without prior acylation. The same result was obtained with GlcNα-PI(C8), confirming that the mannosyltransferase of trypanosomes is divergent from those in yeasts and rodents.

Footnotes

↵* This work was supported by National Institutes of Health Grants GM38545 (to M. A. L.) and GM31278 (to J. R. F.) and Robert Welch Foundation Grants I-1168 (to M. A. L.) and I-782 (to J. R. F.). The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
↵¹ The abbreviations used are:

GPI

glycosylphosphatidylinositol

CHO

Chinese hamster ovary

GlcN-PI

glucosaminyl phosphatidylinositol

MPD

mannose-P-dolichol, PI, phosphatidylinositol

PLC

phospholipase C

HF

hydrogen fluoride

PLD

phospholipase D.
↵² W. T. Doerrler and M. A. Lehrman, unpublished data.
- Received April 15, 1996.
- Revision received August 5, 1996.