Mutational analysis of the karmellae-inducing signal in Hmg1p, a yeast HMG-CoA reductase isozyme

In response to elevated levels of HMG-CoA reductase, an integral endoplasmi c reticulum (ER) membrane protein, cells assemble novel ER arrays. These me mbranes provide useful models for exploration of ER structure and function, as well as general features of membrane biogenesis and turnover, Yeast exp ress two functional HMG-CoA reductase isozymes, Hmg1p and Hmg2p, each of wh ich induces morphologically different ER arrays. Hmg1p induces stacks of pa ired nuclear-associated membranes called karmellae, In contrast, Hmg2p indu ces peripheral ER membrane arrays and short nuclear-associated membrane sta cks. In spite of their ability to induce different cellular responses, both Hmg1p and Hmg2p have similar structures, including a polytopic membrane do main containing eight predicted transmembrane helices, By examining a serie s of recombinant HMG-CoA reductase proteins, our laboratory previously demo nstrated that the last ER-lumenal loop (Loop G) of the Hmg1p membrane domai n contains a signal needed far proper karmellae assembly. Our goal was to e xamine the primary sequence requirements within Loop G that were critical f or proper function of this signal. To this end, me randomly mutagenized the Loop G sequence, expressed the mutagenized Hmg1p in yeast, and screened fo r inability to generate karmellae at wild-type levels, Out of approximately 4000 strains with Loop G mutations, we isolated 57 that mere unable to ind uce wild-type levels of karmellae assembly, Twenty-nine of these mutants co ntained one or more point mutations in the Loop G sequence, including nine single point mutants, four of which had severe defects in karmellae assembl y, Comparison of these mutations to single point mutations that did not aff ect karmellae assembly did not reveal obvious patterns of sequence requirem ents. For example, both conservative and non-conservative changes mere pres ent in both groups and changes that altered the total charge of the Loop G region were observed in both groups. Our hypothesis is that Loop G serves a s a karmellae-inducing signal by mediating protein-protein or protein-lipid interactions and that amino acids revealed by this analysis may be importa nt for maintaining the proper secondary structure needed for these interact ions. Copyright (C) 2000 John Whey & Sons, Ltd.