N-terminal chimeric constructs improve the expression of sarcoplasmic reticulum Ca(2+)-ATPase in yeast.
Wild-type and chimeric constructs comprising rabbit sarcoplasmic reticulum (SR) Ca(2+)-ATPase and the N-terminal cytoplasmic portion of yeast plasma membrane H(+)-ATPase were expressed in yeast under control of a heat-shock regulated promoter. The wild-type ATPase was found predominantly in endoplasmic reticulum (ER) membranes. Addition of the first 88 residues of H(+)-ATPase to the Ca(2+)-ATPase N-terminal end promoted a marked shift in the localization of chimeric H(+)/Ca(2+)-ATPase which accumulated in a light membrane fraction associated with yeast smooth ER. Furthermore, there was a three-fold increase in the overall level of expression of chimeric H(+)/Ca(2+)-ATPase. Similar results were obtained for a chimeric Ca(2+)-ATPase containing a hexahistidine sequence added to its N-terminal end. Both H(+)/Ca(2+)-ATPase and 6xHis-Ca(2+)-ATPase were functional as demonstrated by their ability to form a phosphorylated intermediate and undergo fast turnover. Conversely, a replacement chimera in which the N-terminal end of SR Ca(2+)-ATPase was replaced by the corresponding segment of H(+)-ATPase was not stably expressed in yeast membranes. These results indicate that the N-terminal segment of Ca(2+)-ATPase plays an important role in enzyme assembly and contains structural determinants necessary for ER retention of the ATPase.
Reis
E M
EM
Departamento de Bioquímica, Instituto de Química, Universidade de São Paulo, 05508-900, São Paulo, Brazil.
Kurtenbach
E
E
Ferreira
A R
AR
Biselli
P J
PJ
Slayman
C W
CW
Verjovski-Almeida
S
S
D000818:Animals; D000252:Calcium-Transporting ATPases / Q000096:biosynthesis* / Q000235:genetics; D002462:Cell Membrane / Q000201:enzymology; D005455:Fluorescent Antibody Technique; D015966:Gene Expression Regulation, Fungal; D007425:Intracellular Membranes / Q000201:enzymology; D018613:Microscopy, Confocal; D010766:Phosphorylation; D010957:Plasmids; D011233:Precipitin Tests; D011817:Rabbits; D011993:Recombinant Fusion Proteins / Q000235:genetics; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000235:genetics; D012519:Sarcoplasmic Reticulum / Q000201:enzymology*; D016324:Sequence Tagged Sites
D011993:Recombinant Fusion Proteins; D000252:Calcium-Transporting ATPases