Role of loop L5-6 connecting transmembrane segments M5 and M6 in biogenesis and functioning of yeast Pma1 H+-ATPase
Role of loop L5-6 connecting transmembrane segments M5 and M6 in biogenesis and functioning of yeast Pma1 H+-ATPase
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连接跨膜片段 M5 和 M6 的环 L5-6 在酵母 Pma1 H -ATPase 生物发生和功能中的作用
DOI：
10.1134/s0006297915010046
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发表时间：
2015
期刊：
影响因子：
--
通讯作者：
Ca
中科院分区：
文献类型：
--
作者：
Na;Cu;Ag Mg;Atpase;V. Petrov;Ca
L5-6环是一个短的胞质外拉伸（714-DNSLDID），连接跨膜片段M5和M6，并与片段M4和M8一起形成核心，通过该核心，阳离子被H+-、Ca2+-、K+、Na+-、H+、K+-和其他p2 - atp酶运输。为了研究酵母质膜Pma1 H+- atp酶环内的结构-功能关系，采用了丙氨酸和半胱氨酸扫描诱变技术。Ala和Cys取代最保守的残基（Leu717）导致生物发生完全阻断，阻止酶到达分泌囊泡。在Asp714处替换Ala导致突变体表达减少5倍，丧失活性，而替换Cys则完全阻断了生物发生。替代其他残基不会导致酶活性的丧失。对Asp714和Asp720进行了额外的替换（Asp→Asn/Glu）。在Asp714上进行的替换中，只有D714N部分恢复了突变酶的生物发生和功能。然而，所有取代Asp720的突变酶都有活性。表达的突变体（34-95%的野生型水平）表现出足够高的活性（35-108%），可以进行详细分析。其中一个突变体（I719A） ATP水解和H+转运之间的偶联率降低了3倍；然而，I719C突变与野生型酶几乎没有区别。因此，七个位置中的两个位置的取代严重影响了酶的生物发生和/或功能。综上所述，这些结果表明M5 - M6环残基在蛋白质的稳定性和功能中起着重要的作用，它们可能负责跨膜段M5和M6以及酶的其他结构域的正确排列。这可能对酶的调节也很重要。
The L5-6 loop is a short extracytoplasmic stretch (714-DNSLDID) connecting transmembrane segments M5 and M6 and forming along with segments M4 and M8 the core through which cations are transported by H+-, Ca2+-, K+,Na+-, H+,K+-, and other P2-ATPases. To study structure-function relationships within this loop of the yeast plasma membrane Pma1 H+-ATPase, alanine- and cysteine-scanning mutagenesis has been employed. Ala and Cys substitutions for the most conserved residue (Leu717) led to complete block in biogenesis preventing the enzyme from reaching secretory vesicles. The Ala replacement at Asp714 led to five-fold decrease in the mutant expression and loss of its activity, while the Cys substitution blocked biogenesis completely. Replacements of other residues did not lead to loss of enzymatic activity. Additional replacements were made for Asp714 and Asp720 (Asp→Asn/Glu). Of the substitutions made at Asp714, only D714N partially restored the mutant enzyme biogenesis and functioning. However, all mutant enzymes with substituted Asp720 were active. The expressed mutants (34–95% of the wild-type level) showed activity high enough (35–108%) to be analyzed in detail. One of the mutants (I719A) had three-fold reduced coupling ratio between ATP hydrolysis and H+ transport; however, the I719C mutation was rather indistinguishable from the wild-type enzyme. Thus, substitutions at two of the seven positions seriously affected biogenesis and/or functioning of the enzyme. Taken together, these results suggest that the M5–M6 loop residues play an important role in protein stability and function, and they are probably responsible for proper arrangement of transmembrane segments M5 and M6 and other domains of the enzyme. This might also be important for the regulation of the enzyme.
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DOI：
--
发表时间：
2000
期刊：
The Journal of experimental biology
影响因子：
--
作者：
A. Ambesi;M. Miranda;V. Petrov;C. Slayman
通讯作者：
A. Ambesi;M. Miranda;V. Petrov;C. Slayman
鉴定由 K( )-竞争性光亲和抑制剂标记的 H ,K( )-ATP 酶胞质外区域。
DOI：
--
发表时间：
1991
期刊：
The Journal of biological chemistry
影响因子：
--
作者：
Munson,KB;Gutierrez,C;Balaji,VN;Ramnarayan,K;Sachs,G
通讯作者：
Sachs,G
影响因子：
2.9
作者：
LUTSENKO, S;KAPLAN, JH
通讯作者：
KAPLAN, JH
分泌囊泡中酵母质膜[H]ATP酶的表达。
DOI：
--
发表时间：
1991
期刊：
The Journal of biological chemistry
影响因子：
--
作者：
Nakamoto,RK;Rao,R;Slayman,CW
通讯作者：
Slayman,CW
Na ,K( )-ATPase α 亚基的 M5-M6 发夹的膜配置是配体依赖性的。
DOI：
10.1073/pnas.92.17.7936
发表时间：
1995
影响因子：
11.1
作者：
Lutsenko,S;Anderko,R;Kaplan,JH
通讯作者：
Kaplan,JH