scholarly journals The Rate-limiting Enzyme in Phosphatidylcholine Synthesis Regulates Proliferation of the Nucleoplasmic Reticulum

2005 ◽  
Vol 16 (3) ◽  
pp. 1120-1130 ◽  
Author(s):  
Thomas A. Lagace ◽  
Neale D. Ridgway
Keyword(s):  
Membrane Binding ◽  
Chinese Hamster ◽  
Lipid Synthesis ◽  
Temperature Sensitive ◽  
Direct Role ◽  
Tubule Formation ◽  
Ctp:Phosphocholine Cytidylyltransferase ◽  
Nucleoplasmic Reticulum ◽  
Rate Limiting

The nucleus contains a network of tubular invaginations of the nuclear envelope (NE), termed the nucleoplasmic reticulum (NR), implicated in transport, gene expression, and calcium homeostasis. Here, we show that proliferation of the NR, measured by the frequency of NE invaginations and tubules, is regulated by CTP:phosphocholine cytidylyltransferase-α (CCTα), the nuclear and rate-limiting enzyme in the CDP–choline pathway for phosphatidylcholine (PtdCho) synthesis. In Chinese hamster ovary (CHO)-K1 cells, fatty acids triggered activation and translocation of CCTα onto intranuclear tubules characteristic of the NR. This was accompanied by a twofold increase in NR tubules quantified by immunostaining for lamin A/C or the NE. CHO MT58 cells expressing a temperature-sensitive CCTα allele displayed reduced PtdCho synthesis and CCTα expression and minimal proliferation of the NR in response to oleate compared with CHO MT58 cells stably expressing CCTα. Expression of CCTα mutants in CHO58 cells revealed that both enzyme activity and membrane binding promoted NR proliferation. In support of a direct role for membrane binding in NR tubule formation, recombinant CCTα caused the deformation of liposomes into tubules in vitro. This demonstrates that a key nuclear enzyme in PtdCho synthesis coordinates lipid synthesis and membrane deformation to promote formation of a dynamic nuclear-cytoplasmic interface.

scholarly journals Expansion of the Nucleoplasmic Reticulum Requires the Coordinated Activity of Lamins and CTP:Phosphocholine Cytidylyltransferase α

2008 ◽  
Vol 19 (1) ◽  
pp. 237-247 ◽  
Author(s):  
Karsten Gehrig ◽  
Rosemary B. Cornell ◽  
Neale D. Ridgway
Keyword(s):  
Nuclear Membrane ◽  
Fluorescent Protein ◽  
Membrane Binding ◽  
Membrane Curvature ◽  
Tubule Formation ◽  
Ctp:Phosphocholine Cytidylyltransferase ◽  
Binding Function ◽  
Nucleoplasmic Reticulum ◽  
Green Fluorescent

The nucleoplasmic reticulum (NR), a nuclear membrane network implicated in signaling and transport, is formed by the biosynthetic and membrane curvature-inducing properties of the rate-limiting enzyme in phosphatidylcholine synthesis, CTP:phosphocholine cytidylyltransferase (CCT) α. The NR is formed by invagination of the nuclear envelope and has an underlying lamina that may contribute to membrane tubule formation or stability. In this study we investigated the role of lamins A and B in NR formation in response to expression and activation of endogenous and fluorescent protein-tagged CCTα. Similarly to endogenous CCTα, CCT-green fluorescent protein (GFP) reversibly translocated to nuclear tubules projecting from the NE in response to oleate, a lipid promoter of CCT membrane binding. Coexpression and RNA interference experiments revealed that both CCTα and lamin A and B were necessary for NR proliferation. Expression of CCT-GFP mutants with compromised membrane-binding affinity produced fewer nuclear tubules, indicating that the membrane-binding function of CCTα promotes the expansion of the NR. Proliferation of atypical bundles of nuclear membrane tubules by a CCTα mutant that constitutively associated with membranes revealed that expansion of the double-bilayer NR requires the coordinated assembly of an underlying lamin scaffold and induction of membrane curvature by CCTα.

scholarly journals Mutations affecting the tRNA-splicing endonuclease activity of Saccharomyces cerevisiae.

Genetics ◽  
1988 ◽  
Vol 118 (4) ◽  
pp. 609-617
Author(s):  
M Winey ◽  
M R Culbertson
Keyword(s):  
Growth Defect ◽  
Temperature Sensitive ◽  
Gene Products ◽  
Endonuclease Activity ◽  
Temperature Dependent ◽  
Direct Role ◽  
Trna Splicing

Abstract Two unlinked mutations that alter the enzyme activity of tRNA-splicing endonuclease have been identified in yeast. The sen1-1 mutation, which maps on chromosome 12, causes temperature-sensitive growth, reduced in vitro endonuclease activity, and in vivo accumulation of unspliced pre-tRNAs. The sen2-1 mutation does not confer a detectable growth defect, but causes a temperature-dependent reduction of in vitro endonuclease activity. Pre-tRNAs do not accumulate in sen2-1 strains. The in vitro enzyme activities of sen1-1 and sen2-1 complement in extracts from a heterozygous diploid, but fail to complement in mixed extracts from separate sen1-1 and sen2-1 haploid strains. These results suggest a direct role for SEN gene products in the enzymatic removal of introns from tRNA that is distinct from the role of other products known to affect tRNA splicing.

scholarly journals A role for Yip1p in COPII vesicle biogenesis

2003 ◽  
Vol 163 (1) ◽  
pp. 57-69 ◽  
Author(s):  
Matthew Heidtman ◽  
Catherine Z. Chen ◽  
Ruth N. Collins ◽  
Charles Barlowe
Keyword(s):  
Secretory Pathway ◽  
Protein Transport ◽  
Genetic Interaction ◽  
Temperature Sensitive ◽  
Rab Gtpases ◽  
Direct Role ◽  
Interacting Protein ◽  
Copii Vesicle ◽  
Vesicle Biogenesis

Yeast Ypt1p-interacting protein (Yip1p) belongs to a conserved family of transmembrane proteins that interact with Rab GTPases. We encountered Yip1p as a constituent of ER-derived transport vesicles, leading us to hypothesize a direct role for this protein in transport through the early secretory pathway. Using a cell-free assay that recapitulates protein transport from the ER to the Golgi complex, we find that affinity-purified antibodies directed against the hydrophilic amino terminus of Yip1p potently inhibit transport. Surprisingly, inhibition is specific to the COPII-dependent budding stage. In support of this in vitro observation, strains bearing the temperature-sensitive yip1-4 allele accumulate ER membranes at a nonpermissive temperature, with no apparent accumulation of vesicle intermediates. Genetic interaction analyses of the yip1-4 mutation corroborate a function in ER budding. Finally, ordering experiments show that preincubation of ER membranes with COPII proteins decreases sensitivity to anti-Yip1p antibodies, indicating an early requirement for Yip1p in vesicle formation. We propose that Yip1p has a previously unappreciated role in COPII vesicle biogenesis.

Oxysterol activation of phosphatidylcholine synthesis involves CTP:phosphocholine cytidylyltransferase α translocation to the nuclear envelope

2009 ◽  
Vol 418 (1) ◽  
pp. 209-217 ◽  
Author(s):  
Karsten Gehrig ◽  
Thomas A. Lagace ◽  
Neale D. Ridgway
Keyword(s):  
Nuclear Envelope ◽  
Regulatory Element ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Protein Cleavage ◽  
Ovary Cells ◽  
Ctp:Phosphocholine Cytidylyltransferase ◽  
Indirect Relationship ◽  
Phosphatidylcholine Synthesis

In addition to suppressing cholesterol synthesis and uptake, oxysterols also activate glycerophospholipid and SM (sphingomyelin) synthesis, possibly to buffer cells from excess sterol accumulation. In the present study, we investigated the effects of oxysterols on the CDP-choline pathway for PtdCho (phosphatidylcholine) synthesis using wild-type and sterol-resistant CHO (Chinese-hamster ovary) cells expressing a mutant of SCAP [SREBP (sterol-regulatory-element-binding protein) cleavage-activating protein] (CHO-SCAP D443N). [3H]Choline-labelling experiments showed that 25OH (25-hydroxycholesterol), 22OH (22-hydroxycholesterol) and 27OH (27-hydroxycholesterol) increased PtdCho synthesis in CHO cells as a result of CCTα (CTP:phosphocholine cytidylyltransferase α) translocation and activation at the NE (nuclear envelope). These oxysterols also activate PtdCho synthesis in J774 macrophages. in vitro, CCTα activity was stimulated 2- to 2.5-fold by liposomes containing 5 mol% 25OH, 22OH or 27OH. Inclusion of up to 5 mol% cholesterol did not further activate CCTα. 25OH activated CCTα in CHO-SCAP D443N cells leading to a transient increase in PtdCho synthesis and accumulation of CDP-choline. CCTα translocation to the NE and intranuclear tubules in CHO-SCAP D443N cells was complete after 1 h exposure to 25OH compared with only partial translocation by 4–6 h in CHO-Mock cells. These enhanced responses in CHO-D443N cells were sterol-dependent since depletion with cyclodextrin or lovastatin resulted in reduced sensitivity to 25OH. However, the lack of effect of cholesterol on in vitro CCT activity indicates an indirect relationship or involvement of other sterols or oxysterol. We conclude that translocation and activation of CCTα at nuclear membranes by side-chain hydroxylated sterols are regulated by the cholesterol status of the cell.

scholarly journals Chinese hamster ovary cell mutants with temperature-sensitive defects in endocytosis. I. Loss of function on shifting to the nonpermissive temperature.

1986 ◽  
Vol 103 (6) ◽  
pp. 2283-2297 ◽  
Author(s):  
C F Roff ◽  
R Fuchs ◽  
I Mellman ◽  
A R Robbins
Keyword(s):  
Chinese Hamster Ovary Cell ◽  
Chinese Hamster Ovary ◽  
Chinese Hamster ◽  
Ovary Cell ◽  
Temperature Sensitive ◽  
Permissive Temperature ◽  
Nonpermissive Temperature ◽  
Phenotypic Changes ◽  
Endosomal Acidification

We have isolated three independent Chinese hamster ovary cell mutants (B3853, I223, and M311) with temperature-sensitive, pleiotropic defects in receptor-mediated endocytosis. Activities affected at 41 degrees C include uptake via the D-mannose 6-phosphate receptor, accumulation of Fe from diferric transferrin, uptake of alpha 2-macroglobulin, compartmentalization of newly synthesized acid hydrolases, resistance to ricin, and sensitivity to diphtheria and Pseudomonas toxins and modeccin. The three mutants also displayed decreased sialylation of some secreted glycoproteins at 41 degrees C, reminiscent of the nonconditional mutant DTG1-5-4 that showed both endocytic and Golgi-associated defects (Robbins, A.R., C. Oliver, J.L. Bateman, S.S. Krag, C.J. Galloway, and I. Mellman, 1984, J. Cell Biol., 99:1296-1308). Phenotypic changes were detectable within 30 min after transfer of the mutants to 41 degrees C; maximal alteration of most susceptible functions was obtained 4 h after temperature shift. At 39 degrees C, the mutants exhibited many but not all of the changes manifested at 41 degrees C; resistance to diphtheria and Pseudomonas toxins required the higher temperature. Analysis of cell hybrids showed that B3853 and DTG1-5-4 are in one complementation group ("End1"); M311 and I223 are in another ("End2"). In the End1 mutants, loss of endocytosis correlated with complete loss of ATP-dependent endosomal acidification in vitro; in the End 2 mutants partial loss of acidification was observed. At the nonpermissive temperature, residual levels of endocytic activity in B3853 and M311 were nearly identical; thus, we conclude that the differences measured in endosomal acidification in vitro reflect the different genetic loci affected, rather than the relative severity of the genetic lesions. The mutations in M311 and I223 appear to have different effects on the same protein; in I223 (but not in M311) the full spectrum of phenotypic changes could be produced at the permissive temperature by inhibition of protein synthesis.

Human NADPH-P450 oxidoreductase modulates the level of cytochrome P450 CYP2D6 holoprotein via haem oxygenase-dependent and -independent pathways

2001 ◽  
Vol 356 (2) ◽  
pp. 613-619 ◽  
Author(s):  
Shaohong DING ◽  
Denggao YAO ◽  
Yusuf Y. DEENI ◽  
Brian BURCHELL ◽  
C. Roland WOLF ◽  
...  
Keyword(s):  
Cytochrome P450 ◽  
Cell Line ◽  
Combined Treatment ◽  
Chinese Hamster ◽  
Radical Scavenger ◽  
P450 Oxidoreductase ◽  
Haem Oxygenase ◽  
Mechanistic Basis ◽  
Rate Limiting

NADPH-P450 oxidoreductase (CPR) is essential for the activity of cytochrome P450 (P450). Previous studies demonstrated that CPR regulates the levels of various P450 isoforms in vitro. We investigated the mechanistic basis for this regulation. By transfection of Chinese hamster ovary DUKXB11 cells we obtained the cell line DUKX/2D6, which expressed human CYP2D6, a P450 isoform. Subsequently, DUKX/2D6 cells were transfected with human CPR cDNA to generate the cell line DUKX/2D6/CPR-3. Expression of recombinant CPR decreased the level of spectrally detectable CYP2D6 holoprotein in DUKX/2D6/CPR-3 cells by 70%, whereas the level of immunodetectable apoprotein remained unchanged. Addition of the radical scavenger DMSO increased levels of CYP2D6 holoenzyme in DUKX/2D6/CPR-3 cells but not in DUKX/2D6 cells. A similar effect was noted when cells were grown in the presence of hemin. Importantly, combined treatment with DMSO and hemin increased levels of CYP2D6 holoenzyme in DUKX/2D6/CPR-3 but not in DUKX/2D6 cells even further than either treatment alone. None of these treatments affected the level of immunodetectable CYP2D6. This demonstrates that expression of CPR increases production of damaging radicals but also that CPR may alter haem homoeostasis. In agreement with this, the activity of haem oxygenase, a rate-limiting enzyme in haem metabolism, was compared with that in DUKX/DHFR control cells (expressing dihydrofolate reductase), and was 3-fold higher in DUKX/2D6/CPR-3 but similar in DUKX/2D6 cells. Furthermore, treatment of cells with sodium arsenite increased levels of haem oxygenase concomitant with a marked decrease of spectrally detectable CYP2D6 and a rise in levels of ferritin, which sequesters free iron released from the destruction of haem. These data demonstrate that CPR regulates P450 activity by supplying electrons and also by altering P450 levels via radical-and haem oxygenase-mediated pathways.

scholarly journals Endoprotease PACE4 is Ca2+-dependent and temperature-sensitive and can partly rescue the phenotype of a furin-deficient cell strain

1999 ◽  
Vol 339 (3) ◽  
pp. 639-647 ◽  
Author(s):  
Joseph F. SUCIC ◽  
Joan M. MOEHRING ◽  
Noel M. INOCENCIO ◽  
Jason W. LUCHINI ◽  
Thomas J. MOEHRING
Keyword(s):  
Secretory Pathway ◽  
Protective Antigen ◽  
Chinese Hamster ◽  
Northern Analysis ◽  
Temperature Sensitive ◽  
Exotoxin A ◽  
Wild Type ◽  
Pseudomonas Exotoxin ◽  
Pseudomonas Exotoxin A

PACE4 is a member of the eukaryotic subtilisin-like endoprotease family. The expression of human PACE4 in RPE.40 cells (furin-null mutants derived from Chinese hamster ovary K1 cells) resulted in the rescue of a number of wild-type characteristics, including sensitivity to Sindbis virus and the ability to process the low-density-lipoprotein receptor-related protein. Expression of PACE4 in these cells failed to restore wild-type sensitivity to Pseudomonas exotoxin A. Co-expression of human PACE4 in these cells with either a secreted form of the human insulin pro-receptor or the precursor form of von Willebrand factor resulted in both proproteins being processed; RPE.40 cells were unable to process either precursor protein in the absence of co-expressed PACE4. Northern analysis demonstrated that untransfected RPE.40 cells express mRNA species for four PACE4 isoforms, suggesting that any endogenous PACE4 proteins produced by these cells are either non-functional or sequestered in a compartment outside of the secretory pathway. In experiments in vitro, PACE4 processed diphtheria toxin and anthrax toxin protective antigen, but not Pseudomonas exotoxin A. The activity of PACE4 in vitro was Ca2+-dependent and, unlike furin, was sensitive to temperature changes between 22 and 37 °C. RPE.40 cells stably expressing human PACE4 secreted an endoprotease with the same Ca2+ dependence and temperature sensitivity as that observed in membrane fractions of these cells assayed in vitro. These results, in conjunction with other published work, demonstrate that PACE4 is an endoprotease with more stringent substrate specificity and more limited operating parameters than furin.

scholarly journals PRP4 (RNA4) from Saccharomyces cerevisiae: its gene product is associated with the U4/U6 small nuclear ribonucleoprotein particle.

1989 ◽  
Vol 9 (9) ◽  
pp. 3698-3709 ◽  
Author(s):  
S P Bjørn ◽  
A Soltyk ◽  
J D Beggs ◽  
J D Friesen
Keyword(s):  
Gene Product ◽  
Yeast Cells ◽  
Temperature Sensitive ◽  
Ribonucleoprotein Particle ◽  
Direct Role ◽  
Small Nuclear Ribonucleoprotein ◽  
Cloned Gene ◽  
Nuclear Ribonucleoprotein ◽  
Small Nuclear Ribonucleoprotein Particle

The PRP4 (RNA4) gene product is involved in nuclear mRNA processing in yeast cells; we have previously cloned the gene by complementation of a temperature-sensitive mutation. Sequence and transcript analyses of the cloned gene predicted the gene product to be a 52-kilodalton protein, which was confirmed with antibodies raised against the PRP4 gene product. These antibodies inhibited precursor mRNA splicing in vitro, demonstrating a direct role of PRP4 in splicing. Immunoprecipitations with the antibodies indicated that the PRP4 protein is associated with the U4/U6 small nuclear ribonucleoprotein particle.

scholarly journals FoxO1 Is a Novel Regulator of 20S Proteasome Subunits Expression and Activity

2021 ◽  
Vol 9 ◽  
Author(s):  
Marianna Kapetanou ◽  
Tobias Nespital ◽  
Luke S. Tain ◽  
Andre Pahl ◽  
Linda Partridge ◽  
...  
Keyword(s):  
Proteasome Activity ◽  
20S Proteasome ◽  
Direct Role ◽  
Forkhead Box ◽  
Age Related ◽  
Proteasome Subunits ◽  
Rate Limiting

Proteostasis collapses during aging resulting, among other things, in the accumulation of damaged and aggregated proteins. The proteasome is the main cellular proteolytic system and plays a fundamental role in the maintenance of protein homeostasis. Our previous work has demonstrated that senescence and aging are related to a decline in proteasome content and activities, while its activation extends lifespan in vitro and in vivo in various species. However, the mechanisms underlying this age-related decline of proteasome function and the down-regulation in expression of its subunits remain largely unclear. Here, we demonstrate that the Forkhead box-O1 (FoxO1) transcription factor directly regulates the expression of a 20S proteasome catalytic subunit and, hence, proteasome activity. Specifically, we demonstrate that knockout of FoxO1, but not of FoxO3, in mice severely impairs proteasome activity in several tissues, while depletion of IRS1 enhances proteasome function. Importantly, we show that FoxO1 directly binds on the promoter region of the rate-limiting catalytic β5 proteasome subunit to regulate its expression. In summary, this study reveals the direct role of FoxO factors in the regulation of proteasome function and provides new insight into how FoxOs affect proteostasis and, in turn, longevity.

scholarly journals Acidification of endosome subpopulations in wild-type Chinese hamster ovary cells and temperature-sensitive acidification-defective mutants.

1989 ◽  
Vol 108 (4) ◽  
pp. 1291-1300 ◽  
Author(s):  
S Schmid ◽  
R Fuchs ◽  
M Kielian ◽  
A Helenius ◽  
I Mellman
Keyword(s):  
Chinese Hamster Ovary ◽  
Cho Cells ◽  
Chinese Hamster ◽  
Temperature Sensitive ◽  
Cell Fractionation ◽  
Permissive Temperature ◽  
Wild Type ◽  
Late Endosomes ◽  
Early Endosomes

During endocytosis in Chinese hamster ovary (CHO) cells, Semliki Forest virus (SFV) passes through two distinct subpopulations of endosomes before reaching lysosomes. One subpopulation, defined by cell fractionation using free flow electrophoresis as "early endosomes," constitutes the major site of membrane and receptor recycling; while "late endosomes," an electrophoretically distinct endosome subpopulation, are involved in the delivery of endosomal content to lysosomes. In this paper, the pH-sensitive conformational changes of the SFV E1 spike glycoprotein were used to study the acidification of these defined endosome subpopulations in intact wild-type and acidification-defective CHO cells. Different virus strains were used to measure the kinetics at which internalized SFV was delivered to endosomes of pH less than or equal to 6.2 (the pH at which wild-type E1 becomes resistant to trypsin digestion) vs. endosomes of pH less than or equal to 5.3 (the threshold pH for E1 of the SFV mutant fus-1). By correlating the kinetics of acquisition of E1 trypsin resistance with the transfer of SFV among distinct endosome subpopulations defined by cell fractionation, we found that after a brief residence in vesicles of relatively neutral pH, internalized virus encountered pH less than or equal to 6.2 in early endosomes with a t1/2 of 5 min. Although a fraction of the virus reached a pH of less than or equal to 5.3 in early endosomes, most fus-1 SFV did not exhibit the acid-induced conformational change until arrival in late endosomes (t1/2 = 8-10 min). Thus, acidification of both endosome subpopulations was heterogeneous. However, passage of SFV through a less acidic early endosome subpopulation always preceded arrival in the more acidic late endosome subpopulation. In mutant CHO cells with temperature-sensitive defects in endosome acidification in vitro, acidification of both early and late endosomes was found to be impaired at the restrictive temperature (41 degrees C). The acidification defect was also found to be partially penetrant at the permissive temperature, resulting in the inability of any early endosomes in these cells to attain pH less than or equal to 5.3. In vitro studies of endosomes isolated from mutant cells suggested that the acidification defect is most likely in the proton pump itself. In one mutant, this defect resulted in increased sensitivity of the electrogenic H+ pump to fluctuations in the endosomal membrane potential.

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