Defining the cis-acting requirements in the HMG-CoA reductase gene for karmellae biogenesis

Abstract

In yeast and mammalian cells, increased levels of the ER membrane protein, HMG-CoA reductase (HMGR), induce specific ER membrane arrays. Yeast express two HMGR isozymes, Hmg1p and Hmg2p, each of which induces a morphologically distinct proliferation of the ER. Hmg2p induces short stacks of ER membranes that may be found associated with the nucleus or be present at the cell periphery. Hmg1p induces karmellae, which are stacks of nuclear-associated membranes. The HMGR protein consists of two domains, a polytopic membrane domain at the amino terminus and a cytosolic catalytic domain at the carboxyl terminus. Experiments from our laboratory had indicated that the HMGR membrane domain was exclusively responsible for generation of ER membrane proliferations. We recently discovered that this conclusion is incorrect sequences at the carboxyl terminus can in fact profoundly affect karmellae biogenesis. Specifically, truncations of Hmg1p that removed or shortened the carboxyl terminus were unable to induce karmellae. Our working hypothesis is that a truncated or misfolded cytosolic domain prevents proper signaling for karmellae biogenesis by interfering with the required tertiary structure of the membrane domain.Additionally, our laboratory had previously determined that the last ER lumenal loop (Loop G) of the Hmg1p membrane domain contains a signal needed for proper assembly of karmellae. The folding of Loop G is likely to impact the lumenal interactions of the membrane domain, providing a signal when the conformation of this region permits one. Our goal is to determine the precise amino acid sequence needed for proper function of this signal. To this end, we have randomly mutagenized the Loop G sequence via mutagenic PCR, expressed the mutagenized Hmg1p in yeast, and screened for inability to properly generate karmellae. Analysis of Loop G sequences in karmellae defective as well as karmellae-competent mutants indicates that changes in charged residues of the Loop G region profoundly affect karmellae biogenesis. Our working hypothesis is that Loop G serves as a karmellae-inducing signal by mediating protein-protein interactions and that these charged amino acids may be important for maintaining the proper secondary structure of the Loop G region needed for these interactions.