The carboxy-terminal region of mammalian HSP90 is required for its dimerization and function in vivo

1994 ◽  
Vol 14 (2) ◽  
pp. 1459-1464
Author(s):  
Y Minami ◽  
Y Kimura ◽  
H Kawasaki ◽  
K Suzuki ◽  
I Yahara
Keyword(s):  
Amino Acid ◽  
Terminal Region ◽  
Full Length ◽  
Yeast Cells ◽  
Amino Acid Residues ◽  
Haploid Strain ◽  
Carboxy Terminal Region ◽  
Carboxy Terminal ◽  
Hsp90 Alpha

The majority of mouse HSP90 exists as alpha-alpha and beta-beta homodimers. Truncation of the 15-kDa carboxy-terminal region of mouse HSP90 by digestion with the Ca(2+)-dependent protease m-calpain caused dissociation of the dimer. When expressed in a reticulocyte lysate, the full-length human HSP90 alpha formed a dimeric form. A plasmid harboring human HSP90 alpha cDNA was constructed so that the carboxy-terminal 49 amino acid residues were removed when translated in vitro. This carboxy-terminally truncated human HSP90 alpha was found to exist as a monomer. In contrast, loss of the 118 amino acid residues from the amino terminus of human HSP90 alpha did not affect its in vitro dimerization. Introduction of an expression plasmid harboring the full-length human HSP90 alpha complements the lethality caused by the double mutations of two HSP90-related genes, hsp82 and hsc82, in a haploid strain of Saccharomyces cerevisiae. The carboxy-terminally truncated human HSP90 alpha neither formed dimers in yeast cells nor rescued the lethal double mutant.

scholarly journals The carboxy-terminal region of mammalian HSP90 is required for its dimerization and function in vivo.

1994 ◽  
Vol 14 (2) ◽  
pp. 1459-1464 ◽  
Author(s):  
Y Minami ◽  
Y Kimura ◽  
H Kawasaki ◽  
K Suzuki ◽  
I Yahara
Keyword(s):  
Amino Acid ◽  
Terminal Region ◽  
Full Length ◽  
Yeast Cells ◽  
Amino Acid Residues ◽  
Haploid Strain ◽  
Carboxy Terminal Region ◽  
Carboxy Terminal ◽  
Hsp90 Alpha

The majority of mouse HSP90 exists as alpha-alpha and beta-beta homodimers. Truncation of the 15-kDa carboxy-terminal region of mouse HSP90 by digestion with the Ca(2+)-dependent protease m-calpain caused dissociation of the dimer. When expressed in a reticulocyte lysate, the full-length human HSP90 alpha formed a dimeric form. A plasmid harboring human HSP90 alpha cDNA was constructed so that the carboxy-terminal 49 amino acid residues were removed when translated in vitro. This carboxy-terminally truncated human HSP90 alpha was found to exist as a monomer. In contrast, loss of the 118 amino acid residues from the amino terminus of human HSP90 alpha did not affect its in vitro dimerization. Introduction of an expression plasmid harboring the full-length human HSP90 alpha complements the lethality caused by the double mutations of two HSP90-related genes, hsp82 and hsc82, in a haploid strain of Saccharomyces cerevisiae. The carboxy-terminally truncated human HSP90 alpha neither formed dimers in yeast cells nor rescued the lethal double mutant.

Peroxisome targeting signal of rat liver acyl-coenzyme A oxidase resides at the carboxy terminus

1989 ◽  
Vol 9 (1) ◽  
pp. 83-91
Author(s):  
S Miyazawa ◽  
T Osumi ◽  
T Hashimoto ◽  
K Ohno ◽  
S Miura ◽  
...  
Keyword(s):  
Amino Acid ◽  
Rat Liver ◽  
Coenzyme A ◽  
Terminal Region ◽  
Proteinase K ◽  
Amino Acid Residues ◽  
C Terminus ◽  
Targeting Signal ◽  
Acyl Coenzyme A

To identify the topogenic signal of peroxisomal acyl-coenzyme A oxidase (AOX) of rat liver, we carried out in vitro import experiments with mutant polypeptides of the enzyme. Full-length AOX and polypeptides that were truncated at the N-terminal region were efficiently imported into peroxisomes, as determined by resistance to externally added proteinase K. Polypeptides carrying internal deletions in the C-terminal region exhibited much lower import activities. Polypeptides that were truncated or mutated at the extreme C terminus were totally import negative. When the five amino acid residues at the extreme C terminus were attached to some of the import-negative polypeptides, the import activities were rescued. Moreover, the C-terminal 199 and 70 amino acid residues of AOX directed fusion proteins with two bacterial enzymes to peroxisomes. These results are interpreted to mean that the peroxisome targeting signal of AOX residues at the C terminus and the five or fewer residues at the extreme terminus have an obligatory function in targeting. The C-terminal internal region also has an important role for efficient import, possibly through a conformational effect.

scholarly journals Carboxy-Terminal Region Involved in Activity of Escherichia coli TolC

2001 ◽  
Vol 183 (23) ◽  
pp. 6961-6964 ◽  
Author(s):  
Hiroyasu Yamanaka ◽  
Hiroshi Izawa ◽  
Keinosuke Okamoto
Keyword(s):  
Escherichia Coli ◽  
Amino Acid ◽  
Outer Membrane ◽  
Amino Acid Residue ◽  
Terminal Region ◽  
Transport Activity ◽  
Carboxy Terminus ◽  
Partial Deletion ◽  
Carboxy Terminal Region ◽  
Carboxy Terminal

ABSTRACT The Escherichia coli TolC acts as a channel tunnel in the transport of various molecules across the outer membrane. Partial-deletion studies of tolC revealed that the region extending from the 50th to the 60th amino acid residue from the carboxy terminus plays an important role in this transport activity of TolC.

Characterization of a thioredoxin (BbTrx) from the entomopathogenic fungus Beauveria bassiana and its expression in response to thermal stress

2010 ◽  
Vol 56 (11) ◽  
pp. 934-942 ◽  
Author(s):  
Sheng-Hua Ying ◽  
Xiao-Hui Wang ◽  
Ming-Guang Feng
Keyword(s):  
Thermal Stress ◽  
Amino Acid ◽  
Beauveria Bassiana ◽  
Entomopathogenic Fungus ◽  
Terminal Region ◽  
Reading Frame ◽  
Reduction Activity ◽  
Carboxy Terminal Region ◽  
Carboxy Terminal ◽  
Fungus Beauveria Bassiana

A thioredoxin (BbTrx) was identified from the entomopathogenic fungus Beauveria bassiana . The cloned nucleotide sequence consisted of a 423-bp open reading frame encoding a 141-amino-acid thioredoxin, a 1011-bp 5′ region, and a 419-bp 3′ region. The deduced protein sequence of BbTrx, including a common 95-amino-acid conserved domain and a unique 46-amino-acid carboxy terminal region, was similar (≤38% identity) to that of other thioredoxins and phylogenetically closest to that from Neurospora crassa . In insulin solution containing dithiothreitol at 25 °C, recombinant BbTrx or a truncated form lacking the carboxy terminal region (BbTrxD) exhibited disulfide reduction activity. BbTrxD was more active after pre-incubation at 40–75 °C, and cells expressing BbTrxD showed significantly higher tolerance to thermal stress (51 °C). The BbTrx expression in B. bassiana was greatly elevated when stressed at 40 °C. The results indicate that the new thioredoxin is a potential target for improving the thermotolerance of B. bassiana formulations.

scholarly journals The conserved carboxy-terminal domain of Saccharomyces cerevisiae TFIID is sufficient to support normal cell growth

1991 ◽  
Vol 11 (10) ◽  
pp. 4809-4821
Author(s):  
D Poon ◽  
S Schroeder ◽  
C K Wang ◽  
T Yamamoto ◽  
M Horikoshi ◽  
...  
Keyword(s):  
Amino Acid ◽  
Cell Growth ◽  
Transcription Initiation ◽  
Mutant Form ◽  
Amino Acid Residues ◽  
Wild Type ◽  
Gene Encoding ◽  
Carboxy Terminal Region ◽  
Carboxy Terminal

We have examined the structure-function relationships of TFIID through in vivo complementation tests. A yeast strain was constructed which lacked the chromosomal copy of SPT15, the gene encoding TFIID, and was therefore dependent on a functional plasmid-borne wild-type copy of this gene for viability. By using the plasmid shuffle technique, the plasmid-borne wild-type TFIID gene was replaced with a family of plasmids containing a series of systematically mutated TFIID genes. These various forms of TFIID were expressed from three different promoter contexts of different strengths, and the ability of each mutant form of TFIID to complement our chromosomal TFIID null allele was assessed. We found that the first 61 amino acid residues of TFIID are totally dispensable for vegetative cell growth, since yeast strains containing this deleted form of TFIID grow at wild-type rates. Amino-terminally deleted TFIID was further shown to be able to function normally in vivo by virtue of its ability both to promote accurate transcription initiation from a large number of different genes and to interact efficiently with the Gal4 protein to activate transcription of GAL1 with essentially wild-type kinetics. Any deletion removing sequences from within the conserved carboxy-terminal region of S. cerevisiae TFIID was lethal. Further, the exact sequence of the conserved carboxy-terminal portion of the molecule is critical for function, since of several heterologous TFIID homologs tested, only the highly related Schizosaccharomyces pombe gene could complement our S. cerevisiae TFIID null mutant. Taken together, these data indicate that all important functional domains of TFIID appear to lie in its carboxy-terminal 179 amino acid residues. The significance of these findings regarding TFIID function are discussed.

scholarly journals Basic amino acid residue cluster within nuclear targeting sequence motif is essential for cytoplasmic plectin-vimentin network junctions.

1996 ◽  
Vol 134 (6) ◽  
pp. 1455-1467 ◽  
Author(s):  
B Nikolic ◽  
E Mac Nulty ◽  
B Mir ◽  
G Wiche
Keyword(s):  
Amino Acid ◽  
Intermediate Filaments ◽  
Basic Amino Acid ◽  
Site Directed Mutagenesis ◽  
Sequence Motif ◽  
Amino Acid Residues ◽  
Nuclear Targeting ◽  
Mutant Proteins ◽  
Carboxy Terminal

We have generated a series of plectin deletion and mutagenized cDNA constructs to dissect the functional sequences that mediate plectin's interaction with intermediate filament (IF) networks, and scored their ability to coalign or disrupt intermediate filaments when ectopically expressed in rat kangaroo PtK2 cells. We show that a stretch of approximately 50 amino acid residues within plectin's carboxy-terminal repeat 5 domain serves as a unique binding site for both vimentin and cytokeratin IF networks of PtK2 cells. Part of the IF-binding domain was found to constitute a functional nuclear localization signal (NLS) motif, as demonstrated by nuclear import of cytoplasmic proteins linked to this sequence. Site directed mutagenesis revealed a specific cluster of four basic amino acid residues (arg4277-arg4280) residing within the NLS sequence motif to be essential for IF binding. When mutant proteins corresponding to those expressed in PtK2 cells were expressed in bacteria and purified proteins subjected to a sensitive quantitative overlay binding assay using Eu3+-labeled vimentin, the relative binding capacities of mutant proteins measured were fully consistent with the mutant's phenotypes observed in living cells. Using recombinant proteins we also show by negative staining and rotary shadowing electron microscopy that in vitro assembled vimentin intermediate filaments become packed into dense aggregates upon incubation with plectin repeat 5 domain, in contrast to repeat 4 domain or a mutated repeat 5 domain.

scholarly journals The conserved carboxy-terminal domain of Saccharomyces cerevisiae TFIID is sufficient to support normal cell growth.

1991 ◽  
Vol 11 (10) ◽  
pp. 4809-4821 ◽  
Author(s):  
D Poon ◽  
S Schroeder ◽  
C K Wang ◽  
T Yamamoto ◽  
M Horikoshi ◽  
...  
Keyword(s):  
Amino Acid ◽  
Cell Growth ◽  
Transcription Initiation ◽  
Mutant Form ◽  
Amino Acid Residues ◽  
Wild Type ◽  
Gene Encoding ◽  
Carboxy Terminal Region ◽  
Carboxy Terminal

We have examined the structure-function relationships of TFIID through in vivo complementation tests. A yeast strain was constructed which lacked the chromosomal copy of SPT15, the gene encoding TFIID, and was therefore dependent on a functional plasmid-borne wild-type copy of this gene for viability. By using the plasmid shuffle technique, the plasmid-borne wild-type TFIID gene was replaced with a family of plasmids containing a series of systematically mutated TFIID genes. These various forms of TFIID were expressed from three different promoter contexts of different strengths, and the ability of each mutant form of TFIID to complement our chromosomal TFIID null allele was assessed. We found that the first 61 amino acid residues of TFIID are totally dispensable for vegetative cell growth, since yeast strains containing this deleted form of TFIID grow at wild-type rates. Amino-terminally deleted TFIID was further shown to be able to function normally in vivo by virtue of its ability both to promote accurate transcription initiation from a large number of different genes and to interact efficiently with the Gal4 protein to activate transcription of GAL1 with essentially wild-type kinetics. Any deletion removing sequences from within the conserved carboxy-terminal region of S. cerevisiae TFIID was lethal. Further, the exact sequence of the conserved carboxy-terminal portion of the molecule is critical for function, since of several heterologous TFIID homologs tested, only the highly related Schizosaccharomyces pombe gene could complement our S. cerevisiae TFIID null mutant. Taken together, these data indicate that all important functional domains of TFIID appear to lie in its carboxy-terminal 179 amino acid residues. The significance of these findings regarding TFIID function are discussed.

In vitro phosphorylation of the erythropoietin receptor and an associated protein, pp130

1992 ◽  
Vol 12 (2) ◽  
pp. 706-715
Author(s):  
A Yoshimura ◽  
H F Lodish
Keyword(s):  
Protein Kinase ◽  
Cell Surface ◽  
Terminal Region ◽  
Erythropoietin Receptor ◽  
Protein Kinase Activity ◽  
Carboxy Terminal Region ◽  
Cross Linker ◽  
Carboxy Terminal ◽  
A Site

The cytoplasmic domain of the cloned erythropoietin (EPO) receptor (EPOR) contains no protein kinase motif, yet addition of EPO to EPO-responsive cells causes an increase in protein-tyrosine phosphorylation. Here we show that addition of EPO or interleukin-3 (IL-3) to an IL-3-dependent cell line expressing the wild-type EPOR causes a small fraction (less than 5%) of total cellular EPOR to shift in gel mobility from 66 to 72 kDa, due at least in part to phosphorylation. Using biotinylated EPO as an affinity reagent, we show that the 72-kDa species is greatly enriched on the cell surface. To demonstrate that a protein kinase activity associates with cell surface EPOR, cells were incubated with biotinylated EPO and then cross-linked with a thiol-cleavable chemical cross-linker. The avidin-agarose-selected complexes were incubated with [gamma-32P]ATP. After in vitro phosphorylation and denaturation without reducing agent, both antiphosphotyrosine and anti-EPOR antibodies immunoprecipitated labeled 72-kDa EPOR and an unidentified 130-kDa phosphoprotein (pp130), indicating that a protein kinase is associated with cell surface EPOR and that a fraction of the EPOR was phosphorylated on tyrosine residues either in the cells or during the cell-free phosphorylation reaction. Under reducing conditions, the 72-kDa phosphorylated EPOR but not pp130 was immunoprecipitated with an anti-EPOR antibody, suggesting that the pp130 is bound to the EPOR by the thiol-cleavable chemical cross-linker. Previously, we showed that deletion of the 42 carboxy-terminal amino acids of the EPOR allows cells to grow in 1/10 the normal EPO concentration, without affecting receptor number or affinity. Two carboxy-terminal truncated EPO receptors that are hyperresponsive to EPO were poorly phosphorylated during the in vitro reaction, suggesting that the carboxy-terminal region of the EPOR contains a site for phosphorylation or a site for interaction with a protein kinase. Our data suggests that phosphorylation or interaction with a protein kinase in the carboxy-terminal region may down-modulate the proliferative action of the EPOR.

scholarly journals In vitro phosphorylation of the erythropoietin receptor and an associated protein, pp130.

1992 ◽  
Vol 12 (2) ◽  
pp. 706-715 ◽  
Author(s):  
A Yoshimura ◽  
H F Lodish
Keyword(s):  
Protein Kinase ◽  
Cell Surface ◽  
Terminal Region ◽  
Erythropoietin Receptor ◽  
Protein Kinase Activity ◽  
Carboxy Terminal Region ◽  
Cross Linker ◽  
Carboxy Terminal ◽  
A Site

The cytoplasmic domain of the cloned erythropoietin (EPO) receptor (EPOR) contains no protein kinase motif, yet addition of EPO to EPO-responsive cells causes an increase in protein-tyrosine phosphorylation. Here we show that addition of EPO or interleukin-3 (IL-3) to an IL-3-dependent cell line expressing the wild-type EPOR causes a small fraction (less than 5%) of total cellular EPOR to shift in gel mobility from 66 to 72 kDa, due at least in part to phosphorylation. Using biotinylated EPO as an affinity reagent, we show that the 72-kDa species is greatly enriched on the cell surface. To demonstrate that a protein kinase activity associates with cell surface EPOR, cells were incubated with biotinylated EPO and then cross-linked with a thiol-cleavable chemical cross-linker. The avidin-agarose-selected complexes were incubated with [gamma-32P]ATP. After in vitro phosphorylation and denaturation without reducing agent, both antiphosphotyrosine and anti-EPOR antibodies immunoprecipitated labeled 72-kDa EPOR and an unidentified 130-kDa phosphoprotein (pp130), indicating that a protein kinase is associated with cell surface EPOR and that a fraction of the EPOR was phosphorylated on tyrosine residues either in the cells or during the cell-free phosphorylation reaction. Under reducing conditions, the 72-kDa phosphorylated EPOR but not pp130 was immunoprecipitated with an anti-EPOR antibody, suggesting that the pp130 is bound to the EPOR by the thiol-cleavable chemical cross-linker. Previously, we showed that deletion of the 42 carboxy-terminal amino acids of the EPOR allows cells to grow in 1/10 the normal EPO concentration, without affecting receptor number or affinity. Two carboxy-terminal truncated EPO receptors that are hyperresponsive to EPO were poorly phosphorylated during the in vitro reaction, suggesting that the carboxy-terminal region of the EPOR contains a site for phosphorylation or a site for interaction with a protein kinase. Our data suggests that phosphorylation or interaction with a protein kinase in the carboxy-terminal region may down-modulate the proliferative action of the EPOR.

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