The intracellular targeting and membrane topology of 3-hydroxy-3-methylglutaryl-CoA reductase.

We present evidence that the amino-terminal 39 residue region of 3-hydroxy-3-methylglutaryl- (HMG) CoA reductase, which includes the putative first transmembrane span, is a signal sequence for targeting HMG-CoA reductase to the endoplasmic reticulum. This evidence is based upon fractionation, endoglycosidase-H sensitivity and protease protection assays on an in vitro transcription/translocation system programmed with a mutant cDNA of HMG-CoA reductase that is deleted for sequences coding for all of the putative transmembrane spans except the first. We show that the protein product of this mutant cDNA is associated with microsomes, glycosylated, or protected from proteolysis only in the presence of Signal Recognition Particle. Also, we present evidence for a topological model of HMG-CoA reductase that consists of eight transmembrane spans. This evidence is based upon a concanavalin A binding assay for in vivo glycosylation of an engineered glycosylation site in each of a series of mutants of the fusion protein, HMGal (Skalnik, D. G., Narita, H., Kent, C., and Simoni, R. D. (1988) J. Biol. Chem. 263, 6836-6841). This series of mutants was designed such that for each linker segment between transmembrane spans, a mutant was constructed with an engineered glycosylation site introduced into that linker segment. We show that only the mutants with glycosylation sites in the linker segments between transmembrane spans 1 and 2, 3 and 4, and 5 and 6 are glycosylated. These results support an eight transmembrane span model for the topology of HMG-CoA reductase and are inconsistent with a seven-transmembrane span model.