Mitochondrial phosphate transport. Large scale isolation and characterization of the phosphate transport protein from beef heart mitochondria.

Kolbe, Hanno V. J.; Costello, Diane; Wong, Anna; Lu, Renne Chen; Wohlrab, Hartmut

doi:10.1016/s0021-9258(17)47273-5

Cited by 89 publications

(12 citation statements)

References 43 publications

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“…Comparison with LAML dataset of TCGA cohort suggests that the mitochondria-related gene expression profile in paediatric AML is likely distinct. Elevated expression of genes like SLC25A3, FASTKD1, SDHC, ATP5J, which were observed for the first time in our cohort, are involved in mitochondrial energy metabolism (24)(25)(26). Genes like FASLG, HRK and SNCA, which were observed to be downregulated, also play role in prevention of mitochondrial damage and apoptosis inhibition in melanoma/medulloblastoma cell lines (27)(28)(29).…”

Section: Discussionmentioning

confidence: 65%

Decoding mitochondrial genes in pediatric AML and development of a novel prognostic mitochondrial gene signature

Chaudhary

Ganguly

Palanichamy

et al. 2022

Preprint

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Background: Gene expression profile of mitochondrial-related genes is not well deciphered in pediatric acute myeloid leukaemia (AML). We aimed to identify mitochondria-related differentially expressed genes (DEGs) in pediatric AML with their prognostic significance. Methods: Children with de novo AML were included prospectively between July 2016-December 2019. Transcriptomic profiling was done for a subset of samples, stratified by mtDNA copy number. Top mitochondria-related DEGs were identified and validated by real-time PCR. A prognostic gene signature risk score was formulated using DEGs independently predictive of overall survival (OS) in multivariable analysis. Predictive ability of the risk score was estimated along with external validation in The Tumor Genome Atlas (TCGA) AML dataset. Results: In 143 children with AML, twenty mitochondria-related DEGs were selected for validation, of which 16 were found to be significantly dysregulated. Upregulation of SDHC (p<0.001), CLIC1 (p=0.013) and downregulation of SLC25A29 (p<0.001) were independently predictive of inferior OS, and included for developing prognostic risk score. The risk score model was independently predictive of survival over and above ELN risk categorization (Harrell's c-index: 0.675). High-risk patients (risk score above median) had significantly inferior OS (p<0.001) and event free survival (p<0.001); they were associated with poor-risk cytogenetics (p=0.021), ELN intermediate/poor risk group (p=0.016), absence of RUNX1-RUNX1T1 (p=0.027), and not attaining remission (p=0.016). On external validation, the risk score also predicted OS (p=0.019) in TCGA dataset. Conclusion: We identified and validated mitochondria-related DEGs with prognostic impact in pediatric AML and also developed a novel 3-gene based externally validated gene signature predictive of survival.

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Section: Discussionmentioning

confidence: 65%

Decoding mitochondrial genes in pediatric AML and development of a novel prognostic mitochondrial gene signature

Chaudhary

Ganguly

Palanichamy

et al. 2022

Preprint

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“…No sample was applied to lane 5. Lanes 6 and 7 are human carbonic anhydrase B (HCA) taken up in 20% (v/v) sample buffer (Kolbe et al, 1984) at 0.0125 mg/mL with 5 and 10 pL applied to the gel, respectively. TV-Ethylmaleimide (2 pL, 100 mM) was added to the other samples (20 pL), and after lOmin on ice, 5.5 pL of 100% sample buffer was added.…”

Section: Resultsmentioning

confidence: 99%

Yeast Mitochondrial Phosphate Transport Protein Expressed in Escherichia coli. Site-Directed Mutations at Threonine-43 and at a Similar Location in the Second Tandem Repeat (Isoleucine-141)

Wohlrab¹,

Briggs²

1994

Biochemistry

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Yeast mitochondrial phosphate transport activity has been reconstituted from the import receptor (MIR) expressed as inclusion bodies in Escherichia coli. This result undermines the suggestion [Murakami, H., et al. (1993) Proc. Natl. Acad. Sci. U.S.A. 90, 3358-3362] that the MIR has been misidentified as the phosphate transport protein (PTP). PTP was solubilized with N-lauroylsarcosinate and Triton X-100 and purified with a yield of about 2 mg/L of induced bacterial culture. This PTP, reconstituted in liposomes, catalyzes phosphate uptake with a Vmax [24.5 degrees C, net (zero trans), pHi = 8.0, pHe = 6.8] of 0.61 mmol of phosphate min-1 (mg of PTP)-1 and a Km of 1.30 mM. This Vmax is higher and the Km about the same as that obtained with PTP purified from mitochondria. Replacement of Thr43 and Ile141 by other amino acids results in three types of PTP: (a) 2.5-5.0% Vmax of wild-type PTP (PTPwt) (Thr43Cys; Thr43Ser; Ile141Cys), (b) < 0.1% Vmax (detection limit of assay) of PTPwt (Thr43Ala; Thr43Asp), and (c) proton transport uncoupled from phosphate transport (Ile141Cys). Km changes are not significant. Activity of Thr43Cys confirms results obtained with mitochondrially expressed protein. Thus, yeast PTP requires Thr43 and mammalian PTP the similarly located Cys42 for high transport activity. Thr43 and Ile141 are each situated between two basic residues (LysThrArg vs ArgIleArg). Cys substitutions in either of these positions confer the same high N-ethylmaleimide sensitivity to the yeast PTPwt as displayed by the mammalian PTP.(ABSTRACT TRUNCATED AT 250 WORDS)

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“…Immediately after the transport assay, 30 µL of stock proteoliposomes was added to 500 µL of C e (10 mM Tris, 10 mM PIPES, pH adjusted to 6.8 with KOH) at room temperature and centrifuged in a Beckman TL100 ultracentrifuge for 30 min at (3 × 10 5 )g (90 000 rpm) at 4 °C. To the pellet was added 22.5 µL of the SDS gel electrophoresis sample buffer (11), and duplicate 10 µL samples were applied to the gel. After electrophoresis, the gel was stained with Coomassie Blue, destained, and then silver-stained.…”

Section: Methodsmentioning

confidence: 99%

Replacements of Basic and Hydroxyl Amino Acids Identify Structurally and Functionally Sensitive Regions of the Mitochondrial Phosphate Transport Protein

1999

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The mitochondrial phosphate transport protein (PTP) from the yeast Saccharomyces cerevisiae has been expressed in Escherichia coli, purified, and reconstituted. Basic and hydroxyl residues were replaced to identify structurally and functionally important regions in the protein. Physiologically relevant unidirectional transport from extraliposomal (cytosol) pH 6.8 to intraliposomal (matrix) pH 8.0 was assayed. Replacements that affect transport most dramatically are at Lys42 (matrix end of helix A), Thr79 (helix B), Lys90 (cytosol end of helix B), Arg140 and Arg142 (matrix end of helix C), Lys179 and Lys187 (helix D), Ser232 (helix E), and Arg276 (helix F). The deleterious nature of these mutations was confirmed by the observation that the yeast PTP null mutant transformed with any one of these mutant genes cannot grow or has difficulties growing with glycerol as the primary carbon source. More than 90% of transport activity can be blocked by various mutations without affecting growth on glycerol. Alterations in the structure of the transport protein caused by the mutations were characterized by determining the fraction of PTP incorporated into liposomes during reconstitution. The incorporation of all PTPs (wild type and mutant) into liposomes is 15.5 +/- 8.4 ng of PTP/25 microL and fairly independent of the amount of PTP in the initial reconstitution mix (49-212 ng of PTP/25 microL). Arg159Ala and Lys295Gln show the smallest incorporation of 2.3 +/- 1.6 ng of PTP/25 microL and 2.6 +/- 0.2 ng of PTP/25 microL, respectively. Ser145Ala shows the largest incorporation of 37.0 ng of PTP/25 microL. These three mutants show near wild-type reconstituted transport activity. Two of these three mutations are located in the loop connecting the matrix ends of helices C and D, Ser145 at its N-terminal (the matrix end of helix C) and Arg159 near its center. Lys295 is located at the C-terminal of PTP beyond helix F. These results, together with those from other mutations, suggest that like helix A, the protein segment consisting of the loop connecting helices C and D and helix D as well as the C-terminal of PTP beyond helix F faces the subunit interface of this homodimer. The role of the replacement-sensitive residues in the phosphate or in the coupled proton transport path is discussed.

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Mitochondrial phosphate transport. Large scale isolation and characterization of the phosphate transport protein from beef heart mitochondria.

Cited by 89 publications

References 43 publications

Decoding mitochondrial genes in pediatric AML and development of a novel prognostic mitochondrial gene signature

Decoding mitochondrial genes in pediatric AML and development of a novel prognostic mitochondrial gene signature

Yeast Mitochondrial Phosphate Transport Protein Expressed in Escherichia coli. Site-Directed Mutations at Threonine-43 and at a Similar Location in the Second Tandem Repeat (Isoleucine-141)

Replacements of Basic and Hydroxyl Amino Acids Identify Structurally and Functionally Sensitive Regions of the Mitochondrial Phosphate Transport Protein

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