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

被引:5
作者
Almeida, W. I. [1 ]
Martins, O. B. [1 ]
Carvalho-Alves, P. C. [1 ]
机构
[1] Univ Fed Rio de Janeiro, Ctr Ciencias Saude, Inst Bioquim Med, BR-21941590 Rio De Janeiro, Brazil
来源
BIOCHIMICA ET BIOPHYSICA ACTA-BIOMEMBRANES | 2006年 / 1758卷 / 11期
关键词
P-type; ATPase; oligomerization; yeast; protein structure; self-association;
D O I
10.1016/j.bbamem.2006.08.018
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
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 (LCD Delta h, 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 (K-d=1.9 mu M and 2.9 mu M for LCDh and LCD Delta h, 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 LCD Delta h, 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. (c) 2006 Elsevier B.V All rights reserved.
引用
收藏
页码:1768 / 1776
页数:9
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