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Assembly, maturation, delivery, and functional characterization of C. elegans Na,K‐ATPase α‐subunit (i.e. Eat‐6)
Author(s) -
Gatto Craig,
Otsuka Anthony J,
Yaragatupalli Siddhartha,
Artigas Pablo,
Templeton Christopher L
Publication year - 2009
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.23.1_supplement.798.3
Subject(s) - microbiology and biotechnology , protein subunit , rna interference , biology , caenorhabditis elegans , heterologous expression , rna , chemistry , biochemistry , gene , recombinant dna
Na,K‐ATPase (NKA) is responsible for actively transporting 3Na + (out) and 2K + (in) of most eukaryotic cells. These ionic gradients play a vital role in cell excitability, contractility, and osmotic balance. C. elegans is valuable genetic tool for studying eukaryotic protein maturation and delivery. Thus, in order to investigate further the membrane delivery of NKA we have constructed fluorescently tagged alpha and beta subunits of the worm NKA (i.e. eat‐6 ) for expression in C. elegans . The fluorescently tagged constructs were engineered behind a heat shock promoter so protein expression could be controlled. In addition, to verify the role of the α‐subunit, we performed double‐stranded RNA interference against the α‐subunit in worms containing the α‐YFP fusion protein. We found that RNA interference of endogenous worm NKA‐ β decreased the appearance of α‐YFP at the PM, demonstrating the requirement for βin proper NKA delivery consistent with our previous observations in insect cells. Interestingly, C. elegans Eat‐6 has significant sequence divergence from mammalian NKAs (~50% a.a. similarity) and has received limited functional characterization. Therefore, in addition to endogenous assembly and trafficking determinations, we are also functionally characterizing Eat‐6 via heterologous expression in X. laevis oocytes. Supported by NIH grant #GM061583 to CG and GM060190 to CLT.