Heterologous expression of plant vacuolar pyrophosphatase in yeast demonstrates sufficiency of the substrate-binding subunit for proton transport.

Kim, Eugene J.; Zhen, Rui-Guang; Rea, Philip A.

doi:10.1073/pnas.91.13.6128

Cited by 109 publications

(81 citation statements)

References 18 publications

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“…In direct contrast to the yeast system where the specific activity of H ϩ -PPase in yeast vacuoles was at least twice as low as that in plant vacuoles (15,20), significant overexpression of H ϩ -PPase was achieved with the E. coli system. The specific activity of R-PPase in E. coli IMV was an order of magnitude higher than that in R. rubrum chromatophores, signifying the potential applicability of IMV as a rich source for R-PPase purification.…”

Section: E Coli Versus Yeast As a Host Cell For Hmentioning

confidence: 94%

“…ϩ -PPase ExpressionInitially, Kim et al (15) expressed H ϩ -PPase (from the plant Arabidopsis thaliana) in the yeast S. cerevisiae. Yeast was selected for heterologous expression, because it is a vacuolated host lacking endogenous vacuolar H ϩ -PPase.…”

Section: E Coli Versus Yeast As a Host Cell For Hmentioning

confidence: 99%

“…The R. rubrum H ϩ -PPase (R-PPase) is therefore often referred to as PP i synthase by analogy with ATP synthase (12). Both PPase and proton translocation activities are associated with a single polypeptide of 66 -90 kDa (13)(14)(15), which possibly forms a dimer (16,17). About half of the PPase molecule is embedded in the membrane, as estimated from the 14 -16 transmembrane spans predicted by computer modeling (18,19).…”

mentioning

confidence: 99%

“…Four of these protein orthologs have been heterologously expressed in yeast Saccharomyces cerevisiae in forms capable of both PP i hydrolysis and H ϩ translocation (15,19,20,31). The interest in bacterial H ϩ -PPases significantly increased in 1998, when the gene for R-PPase was cloned and sequenced (32).…”

mentioning

confidence: 99%

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H+-Pyrophosphatase of Rhodospirillum rubrum

Belogurov¹,

Turkina²,

Penttinen³

et al. 2002

Journal of Biological Chemistry

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Section: E Coli Versus Yeast As a Host Cell For Hmentioning

confidence: 94%

Section: E Coli Versus Yeast As a Host Cell For Hmentioning

confidence: 99%

mentioning

confidence: 99%

mentioning

confidence: 99%

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H+-Pyrophosphatase of Rhodospirillum rubrum

Belogurov¹,

Turkina²,

Penttinen³

et al. 2002

Journal of Biological Chemistry

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“…The development of expression systems for plant H ϩ -PPases in yeast Saccharomyces cerevisiae (9) and bacterial H ϩ -PPases in Escherichia coli (18) has paved the way for site-directed mutagenesis of these proton pumps. This approach was successfully employed to identify membrane-embedded charged residues contributing to dicyclohexylcarbodiimide binding in H ϩ -PPases (19), reactive Cys residues (18,20), and those residues that determine the potassium requirement of H ϩ -PPases (16).…”

mentioning

confidence: 99%

Elucidating the Role of Conserved Glutamates in H+-pyrophosphatase of Rhodospirillum rubrum

Malinen

Belogurov

Salminen

et al. 2004

Journal of Biological Chemistry

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H ؉ -pyrophosphatase (H ؉ -PPase) catalyzes pyrophosphate-driven proton transport against the electrochemical potential gradient in various biological membranes. All 50 of the known H ؉ -PPase amino acid sequences contain four invariant glutamate residues. In this study, we use site-directed mutagenesis in conjunction with functional studies to determine the roles of the glutamate residues Glu 197 , Glu 202 , Glu 550 , and Glu 649 in the H ؉ -PPase of Rhodospirillum rubrum (R-PPase). All residues were replaced with Asp and Ala. The resulting eight variant R-PPases were expressed in Escherichia coli and isolated as inner membrane vesicles. All substitutions, except E202A, generated enzymes capable of PP i hydrolysis and PP i -energized proton translocation, indicating that the negative charge of Glu 202 is essential for R-PPase function. The hydrolytic activities of all other PPase variants were impaired at low Mg 2؉ concentrations but were only slightly affected at high Mg 2؉ concentrations, signifying that catalysis proceeds through a three-metal pathway in contrast to wild-type R-PPase, which employs both two-and three-metal pathways. Substitution of Glu 197 , Glu 202 , and Glu 649 resulted in decreased binding affinity for the substrate analogues aminomethylenediphosphonate and methylenediphosphonate, indicating that these residues are involved in substrate binding as ligands for bridging metal ions. Following the substitutions of Glu 550 and Glu 649 , R-PPase was more susceptible to inactivation by the sulfhydryl reagent mersalyl, highlighting a role of these residues in maintaining enzyme tertiary structure. None of the substitutions affected the coupling of PP i hydrolysis to proton transport.

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The Secretory System and Machinery for Protein Targeting

Gal

2018

Annual Plant Reviews Online

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Heterologous expression of plant vacuolar pyrophosphatase in yeast demonstrates sufficiency of the substrate-binding subunit for proton transport.

Cited by 109 publications

References 18 publications

H+-Pyrophosphatase of Rhodospirillum rubrum

H+-Pyrophosphatase of Rhodospirillum rubrum

Elucidating the Role of Conserved Glutamates in H+-pyrophosphatase of Rhodospirillum rubrum

The Secretory System and Machinery for Protein Targeting

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