Shrimp ATP synthase genes complement yeast null mutants for ATP hydrolysis but not synthesis activity.
The aim of this study was to examine the feasibility of employing a yeast functional complementation assay for shrimp genes by using the shrimp mitochondrial F(1)F(0)-ATP synthase enzyme complex as a model. Yeast mutants defective in this complex are typically respiratory-deficient and cannot grow on non-fermentable carbon sources such as glycerol, allowing easy verification of functional complementation by yeast growth on media with them as the only carbon source. We cloned the previous published sequence of ATP2 (coding for ATP synthase β subunit) from the Pacific white shrimp Penaeus vannamei (Pv) and also successfully amplified a novel PvATP3 (coding for the ATP synthase γ subunit). Analysis of the putative amino acid sequence of PvATP3 revealed a significant homology with the ATP synthase γ subunit of crustaceans and insects. Complementation assays were performed using full-length ATP2 and ATP3 as well as a chimeric form of ATP2 containing a leader peptide sequence from yeast and a mature sequence from shrimp. However, the shrimp genes were unable to complement the growth of respective yeast mutants on glycerol medium, even though transcriptional expression of the shrimp genes from plasmid-borne constructs in the transformed yeast cells was confirmed by RT-PCR. Interestingly, both PvATP2 and PvATP3 suppressed the lethality of the yeast F(1) mutants after the elimination of mitochondrial DNA, which suggests the assembly of a functional F(1) complex necessary for the maintenance of membrane potential in the ρ(0) state. These data suggest an incompatibility of the shrimp/yeast chimeric F(1)-ATPase with the stalk and probably also the F(0) sectors of the ATP synthase, which is essential for coupled energy transduction and ATP synthesis.
Department of Biotechnology, Faculty of Science, Mahidol University, Bangkok 10400, Thailand. transkitt@yahoo.com
Kitti
K
Wuthisathid
Kornsunee
K
Phiwsaiya
Xin Jie
XJ
Chen
Saengchan
S
Senapin
Timothy W
TW
Flegel
D000255:Adenosine Triphosphate / Q000096:biosynthesis* / Q000737:chemistry; D000595:Amino Acid Sequence; D000818:Animals; D060829:Arthropod Proteins / Q000096:biosynthesis / Q000235:genetics*; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics / Q000302:isolation & purification; D055786:Gene Knockout Techniques; D005816:Genetic Complementation Test; D006868:Hydrolysis; D025261:Mitochondrial Proton-Translocating ATPases / Q000096:biosynthesis / Q000235:genetics*; D008969:Molecular Sequence Data; D033561:Penaeidae / Q000201:enzymology / Q000235:genetics*; D010802:Phylogeny; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics; D012441:Saccharomyces cerevisiae / Q000235:genetics*; D017422:Sequence Analysis, DNA
D060829:Arthropod Proteins; D018076:DNA, Complementary; D011994:Recombinant Proteins; D000255:Adenosine Triphosphate; D025261:Mitochondrial Proton-Translocating ATPases