CLONING AND EXPRESSION OF AN INSECT CA2-ATPASE FROM HELIOTHIS-VIRESCENS()

A complementary DNA for the Tobacco Budworm, Heliothis virescens, sarc o(endo)plasmic reticulum-type Ca2+-ATPase (HVSERCA) has been cloned an d sequenced. cDNA fragments of adult rabbit fast-twitch muscle Ca2+-AT Pase (SERCA1a) were used as heterologous probes to isolate a partial c DNA clone coding for a protein with high homology to the Ca2+-ATPase f rom Drosophila melanogaster (DRSERCA) and vertebrate ER/SR Ca2+ pumps. The entire cDNA clone contains an ORF encoding a protein of 1000 amin o acids which shares the characteristic motifs of a P-type ATPase. HVS ERCA shares 89% identity with DRSERCA, 80% identity with the Artemia C a2+-ATPase and 72% identity with avian and mammalian SERCAs. An insect Ca2+-ATPase-specific polyclonal antiserum has been raised against a f usion protein containing sequence from the cytoplasmic domain of HVSER CA. Heterologous expression of the insect pump in COS-7 cells has been demonstrated by immunocytochemistry and the reticular pattern of stai ning is consistent with an ER localisation. However, the expressed enz yme from COS-7 cells does not appear to be active. (C) 1998 Elsevier S cience B.V.