The localization and biochemical analysis of Arabidopsis thaliana APYRASE1 through 7
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NTPDases (Apyrases) (EC 18.104.22.168) require divalent cations (Mg2+, Ca2+) for hydrolysis of di- and triphosphate nucleotides, but do not hydrolyze monophosphate nucleotides. They are insensitive to inhibitors of F-type, P-type, and V-type ATPases and are categorized as E-type ATPases. They are grouped in the GDA_CD39 superfamily. Seven NTPDases (AtAPY1-7) have been cloned from Arabidopsis. In this work, AtAPY1 or AtAPY2 tagged with C-terminal green fluorescence protein (GFP) and driven by their respective native promoter displayed Golgi apparatus localization. These GFP constructs can rescue the apy1 apy2 double knockout (apy1 apy2 dKO) successfully, which indicates their accuracy and functionality in localization studies. Furthermore, both AtAPY1 and AtAPY2 can complement the Saccharomyces cerevisiae Golgi-localized GDA1 mutant by rescuing its aberrant protein glycosylation phenotype. The GFP tagged AtAPY1 or AtAPY2 constructs in the apy1 apy2 dKO plants can restore microsomal UDP/GDPase activity in the mutants confirming that they both also have functional competency. Loss-of-function apy1, apy2 and APY1RNAi apy2 mutants showed higher levels of galactose in the cell wall monosaccharide analysis. However, the efficiency of the galactose transport was not altered APY1RNAi apy2 mutants. AtAPY3 through 7 all displayed intracellular localization by transiently expressed C-terminal tagged YFP in the onion epidermal cells. AtAPY3 showed a subcellular localization distinct from the others. Biochemical analyses showed that AtAPY3 prefers to hydrolyze NTP more than NDP. AtAPY4 resides in the cis-Golgi. It has fairly weak NTPDase activity but can still rescue some part of the phenotypic defects in Golgi luminal NTPDases mutants. AtAPY5 is a strong NDPase and has a broad spectrum of substrate preferences. It can fully restore phenotypic defects in Golgi luminal NTPDases in yeast. AtAPY6 and AtAPY7 are ER and Golgi associated. However, the expression of these two enzymes cannot be detected in the Saccharomyces cerevisiae host, which prevents further analysis. Taken together these results reveal that the current seven APYRASE members are intracellulary associated with Golgi/ER or unknown vesicles. They all display typical NTPDase enzyme activities that can hydrolyze di- or triphosphate nucleotides in the cells.