Plasma membrane calcium ATPase （PMCA） plays a critical role in transporting Ca^2＋ out of the cytosol across the plasma membrane which is essential both in keeping intracellular Ca^2＋ homeostasis and in biomineralization. In this paper we cloned and localized a gene encoding PMCA from the pearl oyster Pinctada fucata. This PMCA shares similarity with other published PMCAs within the functional domains. Reverse transcription-polymerase chain reaction analysis shows that it is expressed ubiquitously. Furthermore, in situ hybridization reveals that it is expressed in the inner epithelial cells of the outer fold and in the outer epithelial cells of the middle fold, as well as the edge near the shell, which suggests that PMCA may be involved in calcified layer formation. The identification and characterization of oyster PMCA can help to further understand the structural and functional properties of molluscan PMCA, as well as the mechanism of maintaining Ca^2＋ homeostasis and the mechanism of mineralization in pearl oyster.
In oyster biomineralization, large amounts of calcium are absorbed from external media, transported to the mineralization site, and finally deposited via a matrix-mediated process, All these activities are very energy intensive; therefore, investigations of the energy metabolism pathways of different oyster tissues will facilitate understanding of oyster biomineralization physiology. A full-length cDNA encoding the F1- ATPase beta-subunit （the F1-β-subunit, a major calalytic subunit of F-ATPase） from the pearl oyster （Pinctada fucata） was cloned using the homology strategy with a pair of degenerated primers based on the conserved regions of other animals＇ F1-β-subunit genes. Sequencing and structural analyses showed that the obtained sequence shared high identity with other animals＇ F1-β-subunits, and had a unique phosphorylation site of PKC and CK II on the external surface of the putative protein. Results from semi-quantitative reverse transcription-polymerase chain reaction and in situ hybridization demonstrated this oyster F1-β-subunit mRNA is abundant in the gill and mantle, and distributed widely in the periostracal groove, the outer folder. and the dorsal region of the mantle and in the gill epithelial cells. These tissues were the main regions that participate in biomineralization processes such as calcium uptake, transport, and matrix secretion. The results indicate that tissues involved in biomineralization have stronger energy metabolic processes and that F1-ATPase might play an important role in oyster biomineralization by providing energy transport.
Mitochondrial ATP synthase is responsible for the production of the majority of the cellular ATP, which is composed of two major units： Fo and F1. Although much is known about the active complex （5 subunits （αβγδε））, the role of the α subunit in the catalytic mechanism remains unclear, particularly in bivalve animals. This study first cloned and identified the full-length sequence of the mitochondrial H^＋-ATP synthase α subunit cDNA gene in Pinctada fucata using the reverse transcriptase polymerase chain reaction （RT-PCR） technique. The Pinctada fucata mitochondrial H^＋-ATP synthase α subunit contains 1991 nucleotides, with the translation start site at nt 48 （ATG） and the stop codon at nt 1660 （TAA）, encoding a polypeptide 553 amino acids in length, which shares high similarity to that of other animals （81% identity to fruit fly, 82% to carp, and 83% to humans）. Alignment analysis of the well-conserved amino acid domains in the ATPase α subunit, the call3 signal transduction domain, showed that two residues （Asp^358 and Asn^359） differ from any other ATP synthase α subunit. In situ hybridization analysis was used to reveal the wide-spread distribution of mitochondrial H^＋-ATP synthase in various tissues in Pinctada fucata. This work will help further research on pearl energy metabolism to increase the output and quality of pearls to more efficiently utilize our rich pearl oyster resources.
