Cyclosporin A-binding protein (cyclophilin) of Neurospora crassa. One gene codes for both the cytosolic and mitochondrial forms.

Tropschug, Maximilian; Nicholson, Donald W.; Hartl, F. Ulrich; Köhler, Helmut; Pfanner, Nikolaus; Wächter, Elmar; Neupert, Walter

doi:10.1016/s0021-9258(18)68238-9

Cited by 189 publications

(9 citation statements)

References 58 publications

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“…Since Handschumacher et al (1984) reported fluorescence enhancement for bovine cyclophilin with excitation at 289 nm and emission at 340 nm, readings at 340 Sequence alignment of the tryptophan-containing region of cyclophilin-like proteins from eukaryotes and prokaryotes. Sequences are taken from human (Haendler et al, 1987), bovine (Harding et al, 1986), porcine (Takahashi et al, 1989), mouse (Hasel & Sutcliffe, 1990), hamster , rat (Danielson et al, 1988), Echinococcus granulosus (Lightowlers et al, 1989), Drosophila NinaA (Schneuwly et al, 1989), yeast (Haendler et al, 1989), yeast b (Koser et al, 1990), N. crassa (Tropschug et al, 1988), Salmonella typhimurium (Tran et al, 1990), and E. coli (Kawamukai et al, 1989). nm were also taken (not shown), and the fluorescence changes were parallel to the data reported here.…”

Section: Methodssupporting

confidence: 52%

Human and Escherichia coli cyclophilins: sensitivity to inhibition by the immunosuppressant cyclosporin A correlates with a specific tryptophan residue

Li¹,

Chen²,

Walsh³

1991

Biochemistry

159

124

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The human T-cell protein cyclophilin shows high affinity for and is the proposed target of the major immunosuppressant drug cyclosporin A (CsA). Cyclophilin also has peptidyl prolyl cis-trans isomerase activity that is inhibited by CsA with an IC50 of 6 nM, while by contrast a homologous PPIase from Escherichia coli has been found to be much less sensitive to CsA, shown here to be 500-fold less potent at an IC50 of 3000 nM. This E. coli rotamase lacks the single highly conserved tryptophan residue of eukaryotic cyclophilins, and we show here that mutation of the natural F112 to W112 enhances E. coli rotamase susceptibility to CsA inhibition by 23-fold. Correspondingly, the human W121 mutations to F121 or A121 yield cyclophilins with 75- and 200-fold decreased sensitivity to CsA, while kcat/Km values of rotamase activity in a tetrapeptide assay drop only 2- and 13-fold, respectively. This complementary gain and loss of CsA sensitivity to mutation to or from tryptophan validate the indole side chain as a major determinant in immunosuppressant drug recognition and the separation of PPIase catalytic efficiency from CsA affinity.

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Section: Methodssupporting

confidence: 52%

Human and Escherichia coli cyclophilins: sensitivity to inhibition by the immunosuppressant cyclosporin A correlates with a specific tryptophan residue

Li¹,

Chen²,

Walsh³

1991

Biochemistry

159

124

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“…Treating the left over pellet with 5 mM digitonin allowed the recovery of intracellular proteins, including mitochondrial proteins not identified in the previous fraction but no cytosolic proteins and little tubulins. Extraction with increasing concentration of digitonin has been used for characterizing the various compartments of the mitochondria such as proteins of the outer membrane, of the intermediate space, of the inner membrane and of the matrix. , The majority of non extracted proteins remaining after 5 mM digitonin contain mostly tubulins and proteins possibly associated with them. Indeed, the elongation factor 1-α, ATP dependent DEAD box RNA helicase, pyruvate dehydrogenase E1 β-subunit and 60S ribosomal protein L7a, which were identified in fraction 5 (Table ) are known to bind to tubulin in Arabidopsis .…”

Section: Discussionmentioning

confidence: 99%

Prefractionation by Digitonin Extraction Increases Representation of the Cytosolic and Intracellular Proteome of Leishmania infantum

2006

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Proteome coverage is limited by the dynamic range of proteins present in a sample and often is confined to the analysis of abundant proteins. We have developed a protein prefractionation protocol, based on the differential solubilization of membranes using digitonin, that has allowed an increase in the resolution and depth of comparative proteomic studies. This prefractionation protocol can also be used to infer the subcellular localization of hypothetical proteins as tested experimentally using green fluorescent fusion proteins. The abundant tubulins and associated proteins of the cytoskeleton were removed from the sample using digitonin extraction, hence facilitating the visualization of lower abundance proteins. The digitonin prefractionation protocol was applied for a comparative proteomic analysis of the promastigote and amastigote life cycle stages of Leishmania infantum and has allowed the identification of novel proteins expressed in a stage-specific manner.

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“…However, the existence of the two distinct isoforms was still in question judging from the finding that in N. crassa two mRNA species for the cytosolic and the mitochondrial forms of cyclophilin were transcribed from a single gene and eventually produced an identical molecule by posttranslational processing of the transit signal sequence from the precursor of the mitochondrion targeted protein (Tropschug et al, 1988). In contrast, our current results definitively show that E. coli contains two different genes for PPIase and that the two distinctive forms are expressed in a single cell.…”

Section: Discussionmentioning

confidence: 99%

Two distinct forms of peptidylprolyl-cis-trans-isomerase are expressed separately in periplasmic and cytoplasmic compartments of Escherichia coli cells

Hayano¹,

Takahashi²,

Kato³

et al. 1991

Biochemistry

161

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Peptidylprolyl-cis-trans-isomerase (PPIase) is thought to be essential for protein folding in the cell. Two forms, a and b, of PPIase and their corresponding genes were isolated from Escherichia coli cells. Despite their insensitivity to cyclosporin A (CsA), both amino acid sequences were homologous and related to that of pig cyclophilin, a protein that has PPIase activity sensitive to CsA (Takahashi et al., 1989). PPIase a is found to be identical with the E. coli ORF 190 gene product that was sequenced by Kawamukai et al. (1989) and overexpressed by Liu and Walsh (1990). It is translocated into E. coli periplasmic space with the signal sequence. PPIase b lacks a hydrophobic amino acid stretch which could serve as a signal sequence or a transmembrane domain, and it is detected mainly in the bacterial cytoplasm. These findings indicate that proteins with the ability to assist folding of various polypeptides are located on both sides of the inner membrane. Thus, we propose that the folding of some exported proteins may be catalyzed by the periplasmic proline isomerase and, in turn, that some proteins which have isomerized may not be translocated efficiently.

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Cyclosporin A-binding protein (cyclophilin) of Neurospora crassa. One gene codes for both the cytosolic and mitochondrial forms.

Cited by 189 publications

References 58 publications

Human and Escherichia coli cyclophilins: sensitivity to inhibition by the immunosuppressant cyclosporin A correlates with a specific tryptophan residue

Human and Escherichia coli cyclophilins: sensitivity to inhibition by the immunosuppressant cyclosporin A correlates with a specific tryptophan residue

Prefractionation by Digitonin Extraction Increases Representation of the Cytosolic and Intracellular Proteome of Leishmania infantum

Two distinct forms of peptidylprolyl-cis-trans-isomerase are expressed separately in periplasmic and cytoplasmic compartments of Escherichia coli cells

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