Abstract
Site-specific mutagenesis was used to investigate whether Pro160, Pro195, Pro308, Pro312, Pro803, and Pro812 play essential roles in the function of the sarcoplasmic reticulum Ca2(+)-ATPase. All six prolines were substituted with alanine; and in addition, Pro308 was replaced by glycine and Pro312 by glycine as well as by leucine. Mutant cDNAs were expressed in COS-1 cells, and mutant Ca2(+)-ATPases located in the isolated microsomal fraction were examined with respect to Ca2+ uptake activity, Ca2+ dependence of phosphorylation from ATP, and the kinetic properties of the phosphoenzyme intermediates formed from both ATP and Pi. The enzymatic cycle was little affected by substitution of Pro160, Pro195, and Pro812, which are located in the cytoplasmic domain; but replacement of Pro308, Pro312, and Pro803, in the putative transmembrane helices, had a profound impact on the function of the enzyme. All mutations of Pro308 and Pro803 led to ATPases which were characterized by a reduced affinity for Ca2+. These prolines may therefore be involved in the structure of the high affinity Ca2(+)-binding sites in the enzyme. Substitution of Pro312 with alanine or glycine gave rise to mutants unable to transport Ca2+ even though their apparent affinities for Ca2+ in the phosphorylation reaction with ATP were increased. In these enzymes, the ADP-sensitive phosphoenzyme intermediate was stable for at least 5 min at 0 degrees C, whereas the ADP-insensitive phosphoenzyme intermediate decay at a rate similar to that of the wild type. Thus, the inability to transport Ca2+ could be accounted for by a block of ADP-sensitive to ADP-insensitive phosphoenzyme intermediate conformational transition. In contrast, substitution of Pro312 with leucine gave rise to a mutant enzyme that retained about 7% of the normal Ca2+ transport rate. Phosphoenzyme turnover in this mutant also occurred at a low but significant rate, suggesting that the leucine side chain can substitute to some extent for proline.
Highlights
Functional Consequencesof Proline Mutations in the Cytoplasmic and Transmembrane Sectors of the Ca2'-ATPase of Sarcoplasmic Reticulum*
MutantcDNAs were expressed inCOS-1 cells, The Ca2+-ATPaseof the sarcoplasmic reticulum catalyzes and mutant Ca2+-ATPases located crosomal fraction were examined in the with isolated respect tcoma'+ithe uphill transport of Ca2+from the sarcoplasm tothe membrane lumen at the expense of free energy derived from uptake activity, Ca2+ dependence of phosphorylation the hydrolysis of ATP(Hasselbach, 1964; Martonosiand from ATP,and the kinetic properotifetshe phosphoen- Beeler,1983).A structural model for the Ca2+-ATPase has zyme intermediates formed from both ATP aPnid
The been proposed on the basis of the amino acid sequence deenzymatic cycle was little affected by substitution of ducedfrom the cDNAsequence
Summary
Site-specific Mutagenesis-Oligonucleotide-directesdite-specific mutagenesis was carried out according to Kunkel (1985) as described previously (Maruyama and MacLennan, 1988). Mutations were introduced into short restriction fragments that habdeen excised from the full-length rabbit fast-twitch muscle Ca"-ATPase cDNA clone and inserted into the Bluescriptvector (Stratagene, La Jolla, CA). The fragments used were the following: SmaI (position 442)-KpnI (position 663) for mutation ofPro"' and Pro'"", RarnHI(position R65)-Smnl (position 1600) for Pro"' and Pro"?, and AuaI (position 2:153)-RstElI (position 2716) for Pro":' and Pro"". Mutant clones were selected hy hybridization screening withradiolabeledmut.ant oligonucleotidesaccording tostandard procedures (Maniatis et al 1982). In order to verify thatmutations were appropriateandto ensure that no unwanted mutations or deletions hadoccurred, se-. Ported per enzymatic cycle (Inesi, 198.5) and a site concentration as determined hv the steady-state level ofphosphorylation from ATP.
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