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

1996 ◽  
Vol 7 (5) ◽  
pp. 769-789 ◽  
Author(s):  
A J Koning ◽  
C J Roberts ◽  
R L Wright
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Endoplasmic Reticulum ◽  
Subcellular Localization ◽  
Nuclear Envelope ◽  
Fluorescent Protein ◽  
Endoplasmic Reticulum Membrane ◽  
Hmg Coa Reductase ◽  
Endogenous Expression ◽  
Coa Reductase ◽  
Er Membrane

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, Hmg1p 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 Hmg1p 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 Hmg1p was localized in a pattern that resembled that of Hmg1p when expressed at increased levels. Reflecting their different subcellular distributions, elevated levels of Hmg1p and Hmg2p induced sets of ER membrane proliferations with distinct morphologies. The ER membrane protein, Sec61p, was localized in the membranes induced by both Hmg1p and Hmg2p green fluorescent protein (GFP) fusions. In contrast, the lumenal ER protein, Kar2p, was present in Hmg1p:GFP membranes, but only rarely in Hmg2p:GFP membranes. These results indicated that the membranes synthesized in response to Hmg1p 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.

Genetic and biochemical evaluation of eucaryotic membrane protein topology: multiple transmembrane domains of Saccharomyces cerevisiae 3-hydroxy-3-methylglutaryl coenzyme A reductase

1990 ◽  
Vol 10 (2) ◽  
pp. 672-680
Author(s):  
C Sengstag ◽  
C Stirling ◽  
R Schekman ◽  
J Rine
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Endoplasmic Reticulum ◽  
Coenzyme A ◽  
Transmembrane Domains ◽  
Protein Domain ◽  
Yeast Cells ◽  
Endoplasmic Reticulum Membrane ◽  
Hmg Coa Reductase ◽  
Coa Reductase ◽  
Histidinol Dehydrogenase

Both 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase isozymes of the yeast Saccharomyces cerevisiae are predicted to contain seven membrane-spanning domains. Previous work had established the utility of the histidinol dehydrogenase protein domain, encoded by HIS4C, as a topologically sensitive monitor that can be used to distinguish between the lumen of the endoplasmic reticulum and the cytoplasm. This study directly tested the structural predictions for HMG-CoA reductase by fusing the HIS4C domain to specific sites in the HMG-CoA reductase isozymes. Yeast cells containing the HMG-CoA reductase-histidinol dehydrogenase fusion proteins grew on histidinol-containing medium if the HIS4C domain was present on the cytoplasmic side of the endoplasmic reticulum membrane but not if the HIS4C domain was targeted to the endoplasmic reticulum lumen. Systematic exchanges of transmembrane domains between the isozymes confirmed that both isozymes had equivalent membrane topologies. In general, deletion of an even number of putative transmembrane domains did not interfere with the topology of the protein, but deletion or duplication of an odd number of transmembrane domains inverted the orientation of the protein. The data confirmed the earlier proposed topology for yeast HMG-CoA reductase, demonstrated that the yeast enzymes are core glycosylated, and provided in vivo evidence that the properties of transmembrane domains were, in part, dependent upon their context within the protein.

scholarly journals Genetic and biochemical evaluation of eucaryotic membrane protein topology: multiple transmembrane domains of Saccharomyces cerevisiae 3-hydroxy-3-methylglutaryl coenzyme A reductase.

1990 ◽  
Vol 10 (2) ◽  
pp. 672-680 ◽  
Author(s):  
C Sengstag ◽  
C Stirling ◽  
R Schekman ◽  
J Rine
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Endoplasmic Reticulum ◽  
Coenzyme A ◽  
Transmembrane Domains ◽  
Protein Domain ◽  
Yeast Cells ◽  
Endoplasmic Reticulum Membrane ◽  
Hmg Coa Reductase ◽  
Coa Reductase ◽  
Histidinol Dehydrogenase

Both 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase isozymes of the yeast Saccharomyces cerevisiae are predicted to contain seven membrane-spanning domains. Previous work had established the utility of the histidinol dehydrogenase protein domain, encoded by HIS4C, as a topologically sensitive monitor that can be used to distinguish between the lumen of the endoplasmic reticulum and the cytoplasm. This study directly tested the structural predictions for HMG-CoA reductase by fusing the HIS4C domain to specific sites in the HMG-CoA reductase isozymes. Yeast cells containing the HMG-CoA reductase-histidinol dehydrogenase fusion proteins grew on histidinol-containing medium if the HIS4C domain was present on the cytoplasmic side of the endoplasmic reticulum membrane but not if the HIS4C domain was targeted to the endoplasmic reticulum lumen. Systematic exchanges of transmembrane domains between the isozymes confirmed that both isozymes had equivalent membrane topologies. In general, deletion of an even number of putative transmembrane domains did not interfere with the topology of the protein, but deletion or duplication of an odd number of transmembrane domains inverted the orientation of the protein. The data confirmed the earlier proposed topology for yeast HMG-CoA reductase, demonstrated that the yeast enzymes are core glycosylated, and provided in vivo evidence that the properties of transmembrane domains were, in part, dependent upon their context within the protein.

scholarly journals Biogenesis of the crystalloid endoplasmic reticulum in UT-1 cells: evidence that newly formed endoplasmic reticulum emerges from the nuclear envelope.

1986 ◽  
Vol 102 (6) ◽  
pp. 2158-2168 ◽  
Author(s):  
R K Pathak ◽  
K L Luskey ◽  
R G Anderson
Keyword(s):  
Endoplasmic Reticulum ◽  
Nuclear Envelope ◽  
Nuclear Membrane ◽  
Cholesterol Biosynthesis ◽  
Integral Membrane Protein ◽  
Enzyme Synthesis ◽  
Hmg Coa Reductase ◽  
Outer Nuclear Membrane ◽  
Sequence Of Events ◽  
Coa Reductase

The crystalloid endoplasmic reticulum (ER), a specialized smooth ER of the compactin-resistant UT-1 cell, is composed of multiple membrane tubules packed together in a hexagonal pattern. This membrane contains large amounts of 3-hydroxy-3-methylglutaryl coenzyme A (HMG CoA) reductase, an integral membrane protein that enzymatically regulates endogenous cholesterol biosynthesis. Using morphological and immunocytochemical techniques, we have traced the sequence of events in the biogenesis of this ER when compactin-withdrawn UT-1 cells, which do not have a crystalloid ER, are incubated in the presence of compactin. After 15 h of incubation in the presence of compactin, many cells had profiles of ER cisternae that were juxtaposed to the nuclear envelope and studded with ribosomes on their outer membrane. Both the outer nuclear membrane and the ER membrane contained HMG CoA reductase; however, there was little or no detectable enzyme in rough ER that was free in the cytoplasm. With longer times of incubation in the presence of compactin, these cells had lamellar stacks of smooth ER next to the nuclear envelope that contained HMG CoA reductase. Coordinate with the appearance of the smooth ER, crystalloid ER appeared in the same cell. Often regions of continuity were found between the membrane of the smooth ER and the membrane of the crystalloid ER tubules. These studies suggest that HMG CoA reductase is synthesized along the outer nuclear membrane and in response to increased enzyme synthesis, a membrane emerges from the outer nuclear membrane as smooth ER cisternae, which then transforms into crystalloid ER tubules.

scholarly journals Partial deletion of membrane-bound domain of 3-hydroxy-3-methylglutaryl coenzyme A reductase eliminates sterol-enhanced degradation and prevents formation of crystalloid endoplasmic reticulum.

1987 ◽  
Vol 104 (6) ◽  
pp. 1693-1704 ◽  
Author(s):  
H Jingami ◽  
M S Brown ◽  
J L Goldstein ◽  
R G Anderson ◽  
K L Luskey
Keyword(s):  
Endoplasmic Reticulum ◽  
Half Life ◽  
Coenzyme A ◽  
Catalytic Domain ◽  
Carbohydrate Chain ◽  
Hmg Coa Reductase ◽  
Membrane Bound ◽  
Membrane Spanning ◽  
Coa Reductase ◽  
Er Membrane

3-Hydroxy-3-methylglutaryl coenzyme A (HMG CoA) reductase is anchored to the endoplasmic reticulum (ER) membrane by a hydrophobic NH2-terminal domain that contains seven apparent membrane-spanning regions and a single N-linked carbohydrate chain. The catalytic domain, which includes the COOH-terminal two-thirds of the protein, extends into the cytoplasm. The enzyme is normally degraded with a rapid half-life (2 h), but when cells are depleted of cholesterol, its half-life is prolonged to 11 h. Addition of sterols accelerates degradation by fivefold. To explore the requirements for regulated degradation, we prepared expressible reductase cDNAs from which we either deleted two contiguous membrane-spanning regions (numbers 4 and 5) or abolished the single site for N-linked glycosylation. When expressed in hamster cells after transfection, both enzymes retained catalytic activity. The deletion-bearing enzyme continued to be degraded with a rapid half-life in the presence of sterols, but it no longer was stabilized when sterols were depleted. The glycosylation-minus enzyme was degraded at a normal rate and was stabilized normally by sterol deprivation. When cells were induced to overexpress the deletion-bearing enzyme, they did not incorporate it into neatly arranged crystalloid ER tubules, as occurred with the normal and carbohydrate-minus enzymes. Rather, the deletion-bearing enzyme was incorporated into hypertrophied but disordered sheets of ER membrane. We conclude that the carbohydrate component of HMG CoA reductase is not required for proper subcellular localization or regulated degradation. In contrast, the native structure of the transmembrane component is required to form a normal crystalloid ER and to allow the enzyme to undergo regulated degradation by sterols.

scholarly journals Rtn1p Is Involved in Structuring the Cortical Endoplasmic Reticulum

2006 ◽  
Vol 17 (7) ◽  
pp. 3009-3020 ◽  
Author(s):  
Johan-Owen De Craene ◽  
Jeff Coleman ◽  
Paula Estrada de Martin ◽  
Marc Pypaert ◽  
Scott Anderson ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Nuclear Envelope ◽  
Fluorescent Protein ◽  
Cell Cortex ◽  
Proteomic Screen ◽  
Wide Range ◽  
Membrane Spanning ◽  
Green Fluorescent ◽  
Hydrophilic Loop ◽  
Yeast Mutants

The endoplasmic reticulum (ER) contains both cisternal and reticular elements in one contiguous structure. We identified rtn1Δ in a systematic screen for yeast mutants with altered ER morphology. The ER in rtn1Δ cells is predominantly cisternal rather than reticular, yet the net surface area of ER is not significantly changed. Rtn1-green fluorescent protein (GFP) associates with the reticular ER at the cell cortex and with the tubules that connect the cortical ER to the nuclear envelope, but not with the nuclear envelope itself. Rtn1p overexpression also results in an altered ER structure. Rtn proteins are found on the ER in a wide range of eukaryotes and are defined by two membrane-spanning domains flanking a conserved hydrophilic loop. Our results suggest that Rtn proteins may direct the formation of reticulated ER. We independently identified Rtn1p in a proteomic screen for proteins associated with the exocyst vesicle tethering complex. The conserved hydophilic loop of Rtn1p binds to the exocyst subunit Sec6p. Overexpression of this loop results in a modest accumulation of secretory vesicles, suggesting impaired exocyst function. The interaction of Rtn1p with the exocyst at the bud tip may trigger the formation of a cortical ER network in yeast buds.

scholarly journals Ergosterol interacts with Sey1p to promote atlastin‐mediated endoplasmic reticulum membrane fusion in Saccharomyces cerevisiae

2018 ◽  
Vol 33 (3) ◽  
pp. 3590-3600 ◽  
Author(s):  
Miriam Lee ◽  
Yeojin Moon ◽  
Sanghwa Lee ◽  
Changwook Lee ◽  
Youngsoo Jun
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Endoplasmic Reticulum ◽  
Membrane Fusion ◽  
Endoplasmic Reticulum Membrane

Subcellular Localization of 3-Hydroxy-3-methylglutaryl-CoA Reductase in Saccharomyces cerevisiae

1971 ◽  
Vol 70 (1) ◽  
pp. 175-177 ◽  
Author(s):  
Isamu SHIMIZU ◽  
Jun NAGAI ◽  
Hiroshi HATANAKA ◽  
Eiki SAITO ◽  
Hirohiko KATSUKI
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Subcellular Localization ◽  
Coa Reductase

scholarly journals Agonist-evoked inositol trisphosphate receptor (IP3R) clustering is not dependent on changes in the structure of the endoplasmic reticulum

2006 ◽  
Vol 394 (1) ◽  
pp. 57-66 ◽  
Author(s):  
Mark Chalmers ◽  
Michael J. Schell ◽  
Peter Thorn
Keyword(s):  
Endoplasmic Reticulum ◽  
Protein Interactions ◽  
Cell Activation ◽  
Structural Changes ◽  
Fluorescent Protein ◽  
Cluster Formation ◽  
Yellow Fluorescent Protein ◽  
Resting Cells ◽  
Trisphosphate Receptor ◽  
Er Membrane

The size and number of IP3R (inositol 1,4,5-trisphosphate receptor) clusters located on the surface of the ER (endoplasmic reticulum) is hypothesized to regulate the propagation of Ca2+ waves in cells, but the mechanisms by which the receptors cluster are not understood. Using immunocytochemistry, live-cell imaging and heterologous expression of ER membrane proteins we have investigated IP3R clustering in the basophilic cell line RBL-2H3 following the activation of native cell-surface antigen receptors. IP3R clusters are present in resting cells, and upon receptor stimulation, form larger aggregates. Cluster formation and maintenance required the presence of extracellular Ca2+ in both resting and stimulated cells. Using transfection with a marker of the ER, we found that the ER itself also showed structural changes, leading to an increased number of ‘hotspots’, following antigen stimulation. Surprisingly, however, when we compared the ER hotspots and IP3R clusters, we found them to be distinct. Imaging of YFP (yellow fluorescent protein)–IP3R transfected in to living cells confirmed that IP3R clustering increased upon stimulation. Photobleaching experiments showed that the IP3R occupied a single contiguous ER compartment both before and after stimulation, suggesting a dynamic exchange of IP3R molecules between the clusters and the surrounding ER membrane. It also showed a decrease in the mobile fraction after cell activation, consistent with receptor anchoring within clusters. We conclude that IP3R clustering in RBL-2H3 cells is not simply a reflection of bulk-changes in ER structure, but rather is due to the receptor undergoing homotypic or heterotypic protein–protein interactions in response to agonist stimulation.

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