Different subcellular localization of Saccharomyces cerevisiae HMG-CoA reductase isozymes at elevated levels corresponds to distinct endoplasmic reticulum membrane proliferations.

Abstract

In all eucaryotic cell types analyzed, proliferations of the endoplasmic reticulum (ER) can be induced by increasing the levels of certain integral ER proteins. One of the best characterized of these proteins is HMG-CoA reductase, which catalyzes the rate-limiting step in sterol biosynthesis. We have investigated the subcellular distributions of the two HMG-CoA reductase isozymes in Saccharomyces cerevisiae and the types of ER proliferations that arise in response to elevated levels of each isozyme. At endogenous expression levels, Hmglp and Hmg2p were both primarily localized in the nuclear envelope. However, at increased levels, the isozymes displayed distinct subcellular localization patterns in which each isozyme was predominantly localized in a different region of the ER. Specifically, increased levels of Hmglp were concentrated in the nuclear envelope, whereas increased levels of Hmg2p were concentrated in the peripheral ER. In addition, an Hmg2p chimeric protein containing a 77-amino acid lumenal segment from Hmglp was localized in a pattern that resembled that of Hmglp when expressed at increased levels. Reflecting their different subcellular distributions, elevated levels of Hmglp and Hmg2p induced sets of ER membrane proliferations with distinct morphologies. The ER membrane protein, Sec6lp, was localized in the membranes induced by both Hmglp and Hmg2p green fluorescent protein (GFP) fusions. In contrast, the lumenal ER protein, Kar2p, was present in Hmglp:GFP membranes, but only rarely in Hmg2p:GFP membranes. These results indicated that the membranes synthesized in response to Hmglp and Hmg2p were derived from the ER, but that the membranes were not identical in protein composition. We determined that the different types of ER proliferations were not simply due to quantitative differences in protein amounts or to the different half-lives of the two isozymes. It is possible that the specific distributions of the two yeast HMG-CoA reductase isozymes and their corresponding membrane proliferations may reveal regions of the ER that are specialized for certain branches of the sterol biosynthetic pathway.

Improved deconvolution of very weak confocal signals

“…Gene replacement in Saccharomyces cerevisiae was used to label ER membranes with Hmg1-GFP ( Koning et al , 1996). A confocal Z-stack was captured with line accumulation set to ( A ) 8x or ( B ) 1x.…”

Proliferation of the endoplasmic reticulum occurs normally in cells that lack a functional unfolded protein response

“…(Koning et al, 1993(Koning et al, , 1996Profant et al, 2000;Wright et al, 1988) The lack of a growth assay has slowed searches for genes involved in karmellae assembly, since the only way to detect their presence is by direct microscopic examination. The discovery of a strain background (ire1) in which the presence of karmellae conferred a death phenotype was extremely exciting to us (Cox et al, 1997).…”

“…Expression of HMG1 from CUP1 induces rapid assembly of karmellae, with maximal levels occurring in wild-type cells after 4-6 h of copper addition. However, karmellae are present in wild-type cells even in the absence of copper induction (Koning et al, 1996). To determine the 'time zero' levels of karmellae in this experiment, we observed cells 2 h after the shift to medium without inositol.…”

“…Cells were prefixed in glutaraldehyde, postfixed in potassium permanganate and embedded in Spurr's resin, as previously described (Koning et al, 1996;Wright, 2000). Sections were stained with Reynold's lead citrate (Reynolds, 1963) and examined on a Philips CM100 transmission electron microscope operating at 60 kV.…”

“…In mammals, the ER proliferations form hexagonal arrays of smooth membrane tubules known as crystalloid ER (Anderson et al, 1983;Chin et al, 1982). In yeast, the ER proliferations consist of stacked cisternae known as karmellae, which are closely associated with the nucleus (Wright et al, 1988) or can be present in the cytoplasm (Koning et al, 1996). Karmellae and similar ER proliferations provide a useful experimental system for discovering the molecular mechanisms that enable cells to adjust organelle quantity and quality according to physiological demands.…”