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Role of Loop L5-6 Connecting Transmembrane Segments M5 and M6 in Biogenesis and Functioning of Yeast Pma1 H+-ATPase

V. V. Petrov

Skryabin Institute of Biochemistry and Physiology of Microorganisms, Russian Academy of Sciences, 142290 Pushchino, Russia; E-mail: vpetrov07@gmail.com

Received May 26, 2014; Revision received July 23, 2014
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.
KEY WORDS: yeast, plasma membrane, secretory vesicles, biogenesis, Pma1 H+-ATPase, H+ transport, site-directed mutagenesis

DOI: 10.1134/S0006297915010046