Ultrastructural analysis of crystalloid endoplasmic reticulum in UT-1 cells and its disappearance in response to cholesterol

1983 ◽  
Vol 63 (1) ◽  
pp. 1-20 ◽  
Author(s):  
R.G. Anderson ◽  
L. Orci ◽  
M.S. Brown ◽  
L.M. Garcia-Segura ◽  
J.L. Goldstein
Keyword(s):  
Endoplasmic Reticulum ◽  
Chinese Hamster Ovary Cells ◽  
Membrane Surface ◽  
Freeze Fracture ◽  
Chinese Hamster ◽  
Competitive Inhibitor ◽  
Morphological Evidence ◽  
Hmg Coa Reductase ◽  
Membrane Area ◽  
Coa Reductase

The crystalloid endoplasmic reticulum (ER) consists of hexagonally packed membrane tubules that contain 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMG CoA reductase), an intrinsic membrane protein that catalyses the rate-limiting step in cholesterol synthesis. The crystalloid ER appears in a clone of Chinese hamster ovary cells, designated UT-1, that contain high levels of HMG CoA reductase as a result of growth in the presence of compactin, a competitive inhibitor of the reductase. In the present studies, we have used ultrastructural morphometry to estimate that the crystalloid ER: (1) occupies about 15% of the volume of UT-1 cells; (2) contains 3.4-fold more membrane area than the plasma membrane; and (3) contains less than 700 subunits of HMG CoA reductase per micrometer2 of membrane surface. The crystalloid ER tubules contain 2000 intramembrane particles per micrometer2 with a mean diameter of 10.4 nm, as determined by freeze-fracture. The crystalloid ER membranes are low in cholesterol, as indicated by the small number of filipin-cholesterol complexes in freeze-fracture images after treatment with filipin. The addition of cholesterol or related sterols to UT-1 cells promoted a rapid and stepwise disappearance of the crystalloid ER. Initially, the crystalloid ER fragmented into randomly arranged vesicles and tubules. Subsequently, membrane-bound structures disappeared from the cell so that after incubation with cholesterol for 24–72 h, the cells appeared completely normal. We found no morphological evidence that autophagic vacuoles participate in the degradation. We conclude: (1) that the crystalloid ER is more extensive than necessary merely to support HMG CoA reductase; and (2) that upon exposure to cholesterol the crystalloid ER is degraded by a process that does not involve autophagy.

scholarly journals Conservation of promoter sequence but not complex intron splicing pattern in human and hamster genes for 3-hydroxy-3-methylglutaryl coenzyme A reductase.

1987 ◽  
Vol 7 (5) ◽  
pp. 1881-1893 ◽  
Author(s):  
K L Luskey
Keyword(s):  
Transcription Initiation ◽  
Untranslated Region ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Untranslated Regions ◽  
Hmg Coa Reductase ◽  
Ovary Cells ◽  
Splicing Pattern ◽  
Reductase Gene ◽  
Coa Reductase

Regulation of the expression of 3-hydroxy-3-methyglutaryl coenzyme A (HMG-CoA) reductase is a critical step in controlling cholesterol synthesis. Previous studies in cultured Chinese hamster ovary cells have shown that HMG-CoA reductase is transcribed from a cholesterol-regulated promoter to yield a heterogeneous collection of mRNAs with 5' untranslated regions of 68 to 670 nucleotides in length. Synthesis of these molecules is initiated at multiple sites, and multiple donor sites are used to excise an intron in the 5' untranslated region. In the current paper, I report that human HMG-CoA reductase gene resembles the Chinese hamster gene in having multiple sites of transcription initiation that are subject to suppression by cholesterol. The human gene differs from the hamster gene in that a single donor splice site is used to excise the intron in the 5' untranslated region. All of the resulting RNAs have short 5' untranslated regions of 68 to 100 nucleotides. This difference in the splicing pattern of the first intron is species specific and not a peculiarity of cultured cells in that HMG-CoA reductase mRNAs from Syrian hamster livers resemble those of the cultured Chinese hamster ovary cells. Comparison of the DNA sequences of the HMG-CoA reductase promoters from three different species--humans, Syrian hamsters, and Chinese hamsters--shows a highly conserved region of 179 nucleotides that extends from 220 to 42 nucleotides upstream of the transcription initiation sites. This region is 88% identical between the human and Chinese hamster promoter. When fused to the coding region of the Escherichia coli chloramphenicol acetyltransferase gene, this highly conserved region of the reductase gene directs the cholesterol-regulated expression of chloramphenicol acetyltransferase in transfected hamster cells, further indicating the interspecies conservation of the regulatory elements.

Conservation of promoter sequence but not complex intron splicing pattern in human and hamster genes for 3-hydroxy-3-methylglutaryl coenzyme A reductase

1987 ◽  
Vol 7 (5) ◽  
pp. 1881-1893
Author(s):  
K L Luskey
Keyword(s):  
Transcription Initiation ◽  
Untranslated Region ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Untranslated Regions ◽  
Hmg Coa Reductase ◽  
Ovary Cells ◽  
Splicing Pattern ◽  
Reductase Gene ◽  
Coa Reductase

Regulation of the expression of 3-hydroxy-3-methyglutaryl coenzyme A (HMG-CoA) reductase is a critical step in controlling cholesterol synthesis. Previous studies in cultured Chinese hamster ovary cells have shown that HMG-CoA reductase is transcribed from a cholesterol-regulated promoter to yield a heterogeneous collection of mRNAs with 5' untranslated regions of 68 to 670 nucleotides in length. Synthesis of these molecules is initiated at multiple sites, and multiple donor sites are used to excise an intron in the 5' untranslated region. In the current paper, I report that human HMG-CoA reductase gene resembles the Chinese hamster gene in having multiple sites of transcription initiation that are subject to suppression by cholesterol. The human gene differs from the hamster gene in that a single donor splice site is used to excise the intron in the 5' untranslated region. All of the resulting RNAs have short 5' untranslated regions of 68 to 100 nucleotides. This difference in the splicing pattern of the first intron is species specific and not a peculiarity of cultured cells in that HMG-CoA reductase mRNAs from Syrian hamster livers resemble those of the cultured Chinese hamster ovary cells. Comparison of the DNA sequences of the HMG-CoA reductase promoters from three different species--humans, Syrian hamsters, and Chinese hamsters--shows a highly conserved region of 179 nucleotides that extends from 220 to 42 nucleotides upstream of the transcription initiation sites. This region is 88% identical between the human and Chinese hamster promoter. When fused to the coding region of the Escherichia coli chloramphenicol acetyltransferase gene, this highly conserved region of the reductase gene directs the cholesterol-regulated expression of chloramphenicol acetyltransferase in transfected hamster cells, further indicating the interspecies conservation of the regulatory elements.

scholarly journals Sterol-independent Regulation of 3-Hydroxy-3-methylglutaryl-CoA Reductase by Mevalonate in Chinese Hamster Ovary Cells

1989 ◽  
Vol 264 (19) ◽  
pp. 11044-11052 ◽  
Author(s):  
S R Panini ◽  
R Schnitzer-Polokoff ◽  
T A Spencer ◽  
M Sinensky
Keyword(s):  
Chinese Hamster Ovary ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Ovary Cells ◽  
Coa Reductase

Reduction of endogenous GRP78 levels improves secretion of a heterologous protein in CHO cells

1988 ◽  
Vol 8 (10) ◽  
pp. 4063-4070
Author(s):  
A J Dorner ◽  
M G Krane ◽  
R J Kaufman
Keyword(s):  
Endoplasmic Reticulum ◽  
Chinese Hamster Ovary ◽  
Heterologous Protein ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Glycosylation Site ◽  
Ovary Cells ◽  
Tissue Plasminogen ◽  
Stable Association

GRP78 is localized in the endoplasmic reticulum and associates with improperly folded or underglycosylated proteins. The role of GRP78 in secretion was studied in Chinese hamster ovary cells expressing a tissue plasminogen activator (tPA) variant which lacks potential N-linked glycosylation site sequences because of mutagenesis. The expression of variant tPA resulted in elevated levels of GRP78 and its stable association with tPA. The introduction of antisense GRP78 genes resulted in a two- to threefold reduction in GRP78 levels compared with those of the original cells. Cells with reduced levels of GRP78 secreted two- to threefold-higher levels of tPA activity. tPA expressed in these cells displayed reduced association with GRP78, and a greater proportion was processed to the mature form and secreted. These results demonstrate that reduction of GRP78 level can improve the secretion of an associated protein.

scholarly journals Loss of transcriptional activation of three sterol-regulated genes in mutant hamster cells.

1993 ◽  
Vol 13 (9) ◽  
pp. 5175-5185 ◽  
Author(s):  
M J Evans ◽  
J E Metherall
Keyword(s):  
Transcriptional Regulation ◽  
Cell Lines ◽  
Transcriptional Activation ◽  
Cho Cells ◽  
Mutant Cell ◽  
Chinese Hamster ◽  
Wild Type ◽  
Hmg Coa Reductase ◽  
Low Levels ◽  
Coa Reductase

Cholesterol biosynthesis and uptake are controlled by a classic end product-feedback mechanism whereby elevated cellular sterol levels suppress transcription of the genes encoding 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) synthase, HMG-CoA reductase, and the low-density lipoprotein receptor. The 5'-flanking region of each gene contains a common cis-acting element, designated the sterol regulatory element (SRE), that is required for transcriptional regulation. In this report, we describe mutant Chinese hamster ovary (CHO) cell lines that lack SRE-dependent transcription. Mutant cell lines were isolated on the basis of their ability to survive treatment with amphotericin B, a polyene antibiotic that kills cells by interacting with cholesterol in the plasma membrane. Four mutant lines (SRD-6A, -B, -C, and -D) were found to be cholesterol auxotrophs and demonstrated constitutively low levels of mRNA for all three sterol-regulated genes even under conditions of sterol deprivation. The mutant cell lines were found to be genetically recessive, and all four lines belonged to the same complementation group. When transfected with a plasmid containing a sterol-regulated promoter fused to a bacterial reporter gene, SRD-6B cells demonstrated constitutively low levels of transcription, in contrast to wild-type CHO cells, which increased transcription under conditions of sterol deprivation. Mutation of the SREs in this plasmid prior to transfection reduced the level of expression in wild-type CHO cells deprived of sterols to the level of expression found in SRD-6B cells. The defect in SRD-6 cells is limited to transcriptional regulation, since posttranscriptional mechanisms of sterol-mediated regulation were intact: the cells retained the ability to posttranscriptionally suppress HMG-CoA reductase activity and to stimulate acyl-CoA:cholesterol acyltransferase activity. These results suggest that SRD-6 cells lack a factor required for SRE-dependent transcriptional activation. We contrast these cells with a previously isolated oxysterol-resistant cell line (SRD-2) that lacks a factor required for SRE-dependent transcriptional suppression and propose a model for the role of these genetically defined factors in sterol-mediated transcriptional regulation.

Recognized sequence and conformation in design of linear peptides as a competitive inhibitor for HMG-CoA reductase

2007 ◽  
Vol 20 (3) ◽  
pp. 197-203 ◽  
Author(s):  
Valeriy V. Pak ◽  
Minseon Koo ◽  
Lyubov Yun ◽  
Dae Young Kwon
Keyword(s):  
Competitive Inhibitor ◽  
Hmg Coa Reductase ◽  
Coa Reductase

Increase in membrane cholesterol: A possible trigger for degradation of HMG CoA reductase and crystalloid endoplasmic reticulum in UT-1 cells

Cell ◽  
1984 ◽  
Vol 36 (4) ◽  
pp. 835-845 ◽  
Author(s):  
Lelio Orci ◽  
Michael S. Brown ◽  
Joseph L. Goldstein ◽  
Luis M. Garcia-Segura ◽  
Richard G.W. Anderson
Keyword(s):  
Endoplasmic Reticulum ◽  
Membrane Cholesterol ◽  
Hmg Coa Reductase ◽  
Coa Reductase
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