scholarly journals Modulation ofN-Ethylmaleimide-sensitive Factor Activity upon Amino Acid Deprivation

2005 ◽  
Vol 280 (16) ◽  
pp. 16219-16226 ◽  
Author(s):  
Hagai Shorer ◽  
Nira Amar ◽  
Ari Meerson ◽  
Zvulun Elazar
Keyword(s):  
Amino Acid ◽  
Mammalian Cells ◽  
Protein Targeting ◽  
Fluorescent Protein ◽  
Chinese Hamster Ovary Cells ◽  
Yellow Fluorescent Protein ◽  
Chinese Hamster ◽  
Ovary Cells ◽  
Sensitive Factor ◽  
Vesicular Stomatitis

Adaptation of eukaryotic cells to changing environmental conditions entails rapid regulation of protein targeting and transport to specific organelles. Such adaptation is well exemplified in mammalian cells exposed to nitrogen starvation that are triggered to form and transport autophagosomes to lysosomes, thus constituting an inducible intracellular trafficking pathway. Here we investigated the relationship between the general secretory machinery and the autophagic pathway in Chinese hamster ovary cells grown in the absence of amino acid. Utilizing VSVG-YFP (vesicular stomatitis virus G protein fused to yellow fluorescent protein) and norepinephrine as markers for constitutive and regulated exocytosis, respectively, we found that secretion is attenuated in cells grown in media lacking amino acid. Such decrease in exocytosis stems from partial inhibition ofN-ethylmaleimide-sensitive factor ATPase activity, which in turn causes an accumulation of SNARE complexes at both the Golgi apparatus and the plasma membrane of the starved cells. These findings expose a novel cellular strategy to attenuate secretion of proteins under conditions of limited amino acid supply.

scholarly journals Hydrophobic Regions Adjacent to Transmembrane Domains 1 and 5 Are Important for the Targeting of the 70-kDa Peroxisomal Membrane Protein

2007 ◽  
Vol 282 (46) ◽  
pp. 33831-33844 ◽  
Author(s):  
Yoshinori Kashiwayama ◽  
Kota Asahina ◽  
Masashi Morita ◽  
Tsuneo Imanaka
Keyword(s):  
Amino Acid ◽  
Membrane Protein ◽  
Protein Targeting ◽  
Fluorescent Protein ◽  
Chinese Hamster Ovary Cells ◽  
Intracellular Localization ◽  
Chinese Hamster ◽  
Transmembrane Domains ◽  
Peroxisomal Membrane ◽  
Peroxisomal Membrane Protein

The 70-kDa peroxisomal membrane protein (PMP70) is a major component of peroxisomal membranes. Human PMP70 consists of 659 amino acid residues and has six putative transmembrane domains (TMDs). PMP70 is synthesized on cytoplasmic ribosomes and targeted posttranslationally to peroxisomes by an unidentified peroxisomal membrane protein targeting signal (mPTS). In this study, to examine the mPTS within PMP70 precisely, we expressed various COOH-terminally or NH2-terminally deleted constructs of PMP70 fused with green fluorescent protein (GFP) in Chinese hamster ovary cells and determined their intracellular localization by immunofluorescence. In the COOH-terminally truncated PMP70, PMP70(AA.1-144)-GFP, including TMD1 and TMD2 of PMP70, was still localized to peroxisomes. However, by further removal of TMD2, PMP70(AA.1-124)-GFP lost the targeting ability, and PMP70(TMD2)-GFP did not target to peroxisomes by itself. The substitution of TMD2 in PMP70(AA.1-144)-GFP for TMD4 or TMD6 did not affect the peroxisomal localization, suggesting that PMP70(AA.1-124) contains the mPTS and an additional TMD is required for the insertion into the peroxisomal membrane. In the NH2-terminal 124-amino acid region, PMP70 possesses hydrophobic segments in the region adjacent to TMD1. By the disruption of these hydrophobic motifs by the mutation of L21Q/L22Q/L23Q or I70N/L71Q, PMP70(AA.1-144)-GFP lost targeting efficiency. The NH2-terminally truncated PMP70, GFP-PMP70(AA.263-375), including TMD5 and TMD6, exhibited the peroxisomal localization. PMP70(AA.263-375) also possesses hydrophobic residues (Ile307/Leu308) in the region adjacent to TMD5, which were important for targeting. These results suggest that PMP70 possesses two distinct targeting signals, and hydrophobic regions adjacent to the first TMD of each region are important for targeting.

scholarly journals Steric and not structure-specific factors dictate the endocytic mechanism of glycosylphosphatidylinositol-anchored proteins

2009 ◽  
Vol 186 (4) ◽  
pp. 615-628 ◽  
Author(s):  
Pinkesh Bhagatji ◽  
Rania Leventis ◽  
Jonathan Comeau ◽  
Mohammad Refaei ◽  
John R. Silvius
Keyword(s):  
Mammalian Cells ◽  
Protein Targeting ◽  
Folate Receptor ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Fundamental Property ◽  
Membrane Lipid ◽  
Endocytic Pathway ◽  
Ovary Cells ◽  
Lipid Markers

Diverse glycosylphosphatidylinositol (GPI)-anchored proteins enter mammalian cells via the clathrin- and dynamin-independent, Arf1-regulated GPI-enriched early endosomal compartment/clathrin-independent carrier endocytic pathway. To characterize the determinants of GPI protein targeting to this pathway, we have used fluorescence microscopic analyses to compare the internalization of artificial lipid-anchored proteins, endogenous membrane proteins, and membrane lipid markers in Chinese hamster ovary cells. Soluble proteins, anchored to cell-inserted saturated or unsaturated phosphatidylethanolamine (PE)-polyethyleneglycols (PEGs), closely resemble the GPI-anchored folate receptor but differ markedly from the transferrin receptor, membrane lipid markers, and even protein-free PE-PEGs, both in their distribution in peripheral endocytic vesicles and in the manner in which their endocytic uptake responds to manipulations of cellular Arf1 or dynamin activity. These findings suggest that the distinctive endocytic targeting of GPI proteins requires neither biospecific recognition of their GPI anchors nor affinity for ordered-lipid microdomains but is determined by a more fundamental property, the steric bulk of the lipid-anchored protein.

scholarly journals Divergent effect of mammalian PLCζ in generating Ca2+ oscillations in somatic cells compared with eggs

2011 ◽  
Vol 438 (3) ◽  
pp. 545-553 ◽  
Author(s):  
Sally V. Phillips ◽  
Yuansong Yu ◽  
Andreas Rossbach ◽  
Michail Nomikos ◽  
Vyronia Vassilakopoulou ◽  
...  
Keyword(s):  
Cho Cells ◽  
Fluorescent Protein ◽  
Recombinant Protein Expression ◽  
Chinese Hamster Ovary Cells ◽  
Yellow Fluorescent Protein ◽  
Chinese Hamster ◽  
Cho Cell ◽  
Ovary Cells ◽  
Cell Extracts ◽  
Mammalian Fertilization

Sperm PLCζ (phospholipase Cζ) is a distinct phosphoinositide-specific PLC isoform that is proposed to be the physiological trigger of egg activation and embryo development at mammalian fertilization. Recombinant PLCζ has the ability to trigger Ca2+ oscillations when expressed in eggs, but it is not known how PLCζ activity is regulated in sperm or eggs. In the present study, we have transfected CHO (Chinese-hamster ovary) cells with PLCζ fused with either YFP (yellow fluorescent protein) or luciferase and found that PLCζ-transfected cells did not display cytoplasmic Ca2+ oscillations any differently from control cells. PLCζ expression was not associated with changes in CHO cell resting Ca2+ levels, nor with a significantly changed Ca2+ response to extracellular ATP compared with control cells transfected with either YFP alone, a catalytically inactive PLCζ or luciferase alone. Sperm extracts containing PLCζ also failed to cause Ca2+ oscillations in CHO cells. Despite these findings, PLCζ-transfected CHO cell extracts exhibited high recombinant protein expression and PLC activity. Furthermore, either PLCζ-transfected CHO cells or derived cell extracts could specifically cause cytoplasmic Ca2+ oscillations when microinjected into mouse eggs. These data suggest that PLCζ-mediated Ca2+ oscillations may require specific factors that are only present within the egg cytoplasm or be inhibited by factors present only in somatic cell lines.

scholarly journals Functional analysis of Chinese hamster phosphatidylserine synthase 1 through systematic alanine mutagenesis

2004 ◽  
Vol 381 (3) ◽  
pp. 853-859 ◽  
Author(s):  
Tomoko OHSAWA ◽  
Masahiro NISHIJIMA ◽  
Osamu KUGE
Keyword(s):  
Amino Acid ◽  
Chinese Hamster Ovary ◽  
Mammalian Cells ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Amino Acid Sequences ◽  
Amino Acid Residues ◽  
Intact Cells ◽  
Base Exchange ◽  
Ovary Cells

PtdSer (phosphatidylserine) synthesis in mammalian cells occurs through the exchange of L-serine with the base moieties of phosphatidylcholine and phosphatidylethanolamine, which is catalysed by PSS (PtdSer synthase) 1 and 2 respectively. PtdSer synthesis in intact cells and an isolated membrane fraction was inhibited by exogenous PtdSer, indicating that feedback control is involved in the regulation of PtdSer biosynthesis. PSS 1 and 2 are similar in amino acid sequence, with an identity of 32%; however, due to a lack of homology with other known enzymes, their amino acid sequences do not provide information on their catalytic and regulatory mechanisms. In the present study, to identify amino acid residues crucial for the activity and/or regulation of PSS 1, we systematically introduced mutations into a Chinese hamster PSS 1 cDNA clone; namely, each of the 66 polar amino acid residues common to PSS 2 was replaced with an alanine residue. On analysis of Chinese hamster ovary cells transfected with each of the alanine mutant clones, we identified eight amino acid residues (His-172, Glu-197, Glu-200, Asn-209, Glu-212, Asp-216, Asp-221 and Asn-226) as those crucial for the enzyme reaction or the maintenance of the correct structure required for serine base-exchange activity. Among these residues, Asn-209 was suggested to be involved in the recognition and/or binding of free L-serine. We also identified six amino acid residues (Arg-95, His-97, Cys-189, Arg-262, Gln-266 and Arg-336) as those important for regulation of PSS 1. In addition, we found that the alanine mutations at Tyr-111, Asp-166, Arg-184, Arg-323, and Glu-364 affected the production and/or stability of PSS 1 in Chinese hamster ovary cells.

scholarly journals Cell cycle-dependent effects on deoxyribonucleotide and DNA labeling by nucleoside precursors in mammalian cells.

1987 ◽  
Vol 7 (1) ◽  
pp. 532-534 ◽  
Author(s):  
J M Leeds ◽  
C K Mathews
Keyword(s):  
Cell Cycle ◽  
Mammalian Cells ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
G1 Phase ◽  
Ovary Cells ◽  
Dna Labeling ◽  
Metabolic Pool ◽  
Specific Activities ◽  
Cycle Dependent

dCTP pools equilibrated to equivalent specific activities in Chinese hamster ovary cells or in nuclei after incubation of cells with radiolabeled nucleosides, indicating that dCTP in nuclei does not constitute a distinct metabolic pool. In the G1 phase, [5-3H]deoxycytidine labeled dCTP to unexpectedly high specific activities. This may explain reports of replication-excluded DNA precursor pools.

scholarly journals A fluorescent lipid analogue can be used to monitor secretory activity and for isolation of mammalian secretion mutants.

1995 ◽  
Vol 6 (2) ◽  
pp. 135-150 ◽  
Author(s):  
N T Ktistakis ◽  
C Y Kao ◽  
R H Wang ◽  
M G Roth
Keyword(s):  
Chinese Hamster Ovary ◽  
Mammalian Cells ◽  
Secretory Pathway ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Brefeldin A ◽  
Secretory Activity ◽  
Specific Marker ◽  
Ovary Cells ◽  
Fluorescent Lipid

The use of reporter proteins to study the regulation of secretion has often been complicated by posttranslational processing events that influence the secretion of certain proteins, but are not part of the cellular mechanisms that specifically regulate secretion. This has been a particular limitation for the isolation of mammalian secretion mutants, which has typically been a slow process. To provide a reporter of secretory activity independent of protein processing events, cells were labeled with the fluorescent lipid analogue C5-DMB-ceramide (ceramide coupled to the fluorophore boron dipyrromethene difluoride) and its secretion was followed by fluorescence microscopy and fluorescence-activated cell sorting. Brefeldin A, which severely inhibits secretion in Chinese hamster ovary cells, blocked secretion of C5-DMB-ceramide. At high temperature, export of C5-DMB-ceramide was inhibited in HRP-1 cells, which have a conditional defect in secretion. Using C5-DMB-ceramide as a reporter of secretory activity, several different pulse-chase protocols were designed that selected mutant Chinese hamster ovary cells that were resistant to the drug brefeldin A and others that were defective in the transport of glycoproteins to the cell surface. Mutant cells of either type were identified in a mutagenized population at a frequency of 10(-6). Thus, the fluorescent lipid C5-DMB-ceramide can be used as a specific marker of secretory activity, providing an efficient, general approach for isolating mammalian cells with defects in the secretory pathway.

Spontaneous splicing mutations at the dihydrofolate reductase locus in Chinese hamster ovary cells

1986 ◽  
Vol 6 (6) ◽  
pp. 1926-1935
Author(s):  
P J Mitchell ◽  
G Urlaub ◽  
L Chasin
Keyword(s):  
Amino Acid ◽  
Dihydrofolate Reductase ◽  
Splice Site ◽  
Chinese Hamster Ovary ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Splice Sites ◽  
Ovary Cells ◽  
Splicing Mutations ◽  
Intron 4

We isolated and characterized three spontaneous mutants of Chinese hamster ovary cells that were deficient in dihydrofolate reductase activity. All three mutants contained no detectable enzyme activity and produced dihydrofolate reductase mRNA species that were shorter than those of the wild type by about 120 bases. Six exons are normally represented in this mRNA; exon 5 was missing in all three mutant mRNAs. Nuclease S1 analysis of the three mutants indicated that during the processing of the mutant RNA, exon 4 was spliced to exon 6. The three mutant genes were cloned, and the regions around exons 4 and 5 were sequenced. In one mutant, the GT dinucleotide at the 5' end of intron 5 had changed to CT. In a second mutant, the first base in exon 5 had changed from G to T. In a revertant of this mutant, this base was further mutated to A, a return to a purine. Approximately 25% of the mRNA molecules in the revertant were spliced correctly to produce an enzyme with one presumed amino acid change. In the third mutant, the AG at the 3' end of intron 4 had changed to AA. A mutation that partially reversed the mutant phenotype had changed the dinucleotide at the 5' end of intron 4 from GT to AT. The splicing pattern in this revertant was consistent with the use of cryptic donor and acceptor splice sites close to the original sites to produce an mRNA with three base changes and a protein with two amino acid changes. These mutations argue against a scanning model for the selection of splice site pairs and suggest that only a single splice site need be inactivated to bring about efficient exon skipping (a regulatory mechanism for some genes). The fact that all three mutants analyzed exhibited exon 5 splicing mutations indicates that these splice sites are hot spots for spontaneous mutation.

Further Evidence That the Majority of Primary Nuclear RNA Transcripts in Mammalian Cells Do Not Contribute to mRNA

1982 ◽  
Vol 2 (6) ◽  
pp. 701-707
Author(s):  
M. Salditt-Georgieff ◽  
J. E. Darnell
Keyword(s):  
Mammalian Cells ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Cdna Clones ◽  
Ovary Cells ◽  
Rna Molecules ◽  
Rna Transcripts ◽  
Polyadenylic Acid ◽  
Nuclear Molecules ◽  
Nuclear Rna

Nuclear RNA from Chinese hamster ovary cells was effectively separated into polyadenylic acid [poly(A)]-containing [poly (A) + ] and non-poly(A)-containing [poly(A) − ] fractions so that ∼90% of the poly(A) was present in the (A) + fraction. Only 25% of the 5′-terminal caps of the large nuclear molecules were present in the (A) + class, but about 70% of the specific mRNA sequences (assayed with cDNA clones) were in the (A) + class. It appears that many long capped heterogeneous nuclear RNA molecules are of a different sequence category from those molecules that are successfully processed into mRNA.

scholarly journals Molecular Cloning of a Functional Allatostatin Gut/Brain Receptor and an Allatostatin Preprohormone from the SilkwormBombyx mori

2001 ◽  
Vol 276 (50) ◽  
pp. 47052-47060 ◽  
Author(s):  
Thomas Secher ◽  
Camilla Lenz ◽  
Giuseppe Cazzamali ◽  
Gunnar Sørensen ◽  
Michael Williamson ◽  
...  
Keyword(s):  
Amino Acid ◽  
Molecular Cloning ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Peptide Hormone ◽  
Functional Expression ◽  
Bar Gene ◽  
Ovary Cells ◽  
Intron Phasing ◽  
Insect Gut

The cockroach-type or A-type allatostatins are inhibitory insect neuropeptides with the C-terminal sequence Tyr/Phe-X-Phe-Gly-Leu-NH2. Here, we have cloned an A-type allatostatin receptor from the silkwormBombyx mori(BAR). BAR is 361 amino acid residues long, has seven transmembrane domains, shows 60% amino acid residue identity with the firstDrosophilaallatostatin receptor (DAR-1), and 48% identity with the secondDrosophilaallatostatin receptor (DAR-2). The BAR gene has two introns and three exons. These two introns coincide with and have the same intron phasing as two introns in the DAR-1 and DAR-2 genes, showing that the three receptors are not only structurally but also evolutionarily related. Furthermore, we have cloned aBombyxallatostatin preprohormone that contains eight different A-type allatostatins. Chinese hamster ovary cells permanently transfected with BAR DNA react on the addition of 4 × 10−9mBombyxA-type allatostatins with a second messenger cascade (measured as bioluminescence), showing that BAR is a functional A-type allatostatin receptor. Southern blots suggest thatBombyxhas at least one other BAR-related gene in addition to the BAR gene described in this paper. Northern blots and quantitative reverse transcriptase-polymerase chain reaction of different larval tissues show that BAR mRNA is mainly expressed in the gut and to a much lesser extent in the brain. To our knowledge, this is the first report on the molecular cloning and functional expression of an insect gut/brain peptide hormone receptor.

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