Self-association of isolated large cytoplasmic domain of plasma membrane H+ -ATPase from Saccharomyces cerevisiae: role of the phosphorylation domain in a general dimeric model for P-ATPases.
Large cytoplasmic domain (LCD) plasma membrane H+ -ATPase from S. cerevisiae was expressed as two fusion polypeptides in E. coli: a DNA sequence coding for Leu353-Ileu674 (LCDh), comprising both nucleotide (N) and phosphorylation (P) domains, and a DNA sequence coding for Leu353-Thr543 (LCDDeltah, lacking the C-terminus of P domain), were inserted in expression vectors pDEST-17, yielding the respective recombinant plasmids. Overexpressed fusion polypeptides were solubilized with 6 M urea and purified on affinity columns, and urea was removed by dialysis. Their predicted secondary structure contents were confirmed by CD spectra. In addition, both recombinant polypeptides exhibited high-affinity 2',3'-O-(2,4,6-trinitrophenyl)adenosine-5'-triphosphate (TNP-ATP) binding (Kd = 1.9 microM and 2.9 microM for LCDh and LCDDeltah, respectively), suggesting that they have native-like folding. The gel filtration profile (HPLC) of purified LCDh showed two main peaks, with molecular weights of 95 kDa and 39 kDa, compatible with dimeric and monomeric forms, respectively. However, a single elution peak was observed for purified LCDDeltah, with an estimated molecular weight of 29 kDa, as expected for a monomer. Together, these data suggest that LCDh exist in monomer-dimer equilibrium, and that the C-terminus of P domain is necessary for self-association. We propose that such association is due to interaction between vicinal P domains, which may be of functional relevance for H+ -ATPase in native membranes. We discuss a general dimeric model for P-ATPases with interacting P domains, based on published crystallography and cryo-electron microscopy evidence.
Almeida
W I
WI
Instituto de Bioquímica Médica, Centro de Ciências da Saúde, Universidade Federal do Rio de Janeiro, Rio de Janeiro, 21941-590, Brazil. welington@bioqmed.ufrj.br
Martins
O B
OB
Carvalho-Alves
P C
PC
D000255:Adenosine Triphosphate / Q000031:analogs & derivatives; D000595:Amino Acid Sequence; D001483:Base Sequence; D002462:Cell Membrane / Q000201:enzymology*; D003062:Codon; D003593:Cytoplasm / Q000201:enzymology*; D004271:DNA, Fungal / Q000235:genetics / Q000378:metabolism; D019281:Dimerization; D007532:Isoleucine / Q000235:genetics; D007700:Kinetics; D007930:Leucine / Q000235:genetics; D008954:Models, Biological; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010766:Phosphorylation; D017510:Protein Folding; D006180:Proton-Translocating ATPases / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000235:genetics
D003062:Codon; D004271:DNA, Fungal; D011994:Recombinant Proteins; D007532:Isoleucine; C045674:2',3'-O-(2,4,6-trinitro-cyclohexadienylidine)adenosine 5'-triphosphate; D000255:Adenosine Triphosphate; D006180:Proton-Translocating ATPases; D007930:Leucine