SCANNING MUTAGENESIS REVEALS A SIMILAR PATTERN OF MUTATION SENSITIVITY IN TRANSMEMBRANE SEQUENCES M4, M5, AND M6, BUT NOT IN M8, OF THE CA2-ATPASE OF SARCOPLASMIC-RETICULUM (SERCA1A)()

Scanning mutagenesis was performed on all amino acids in transmembrane sequences M5, M6, and M8, which, together with M4, make up the Ca2+ b inding domain of the Ca2+-ATPase of sarcoplasmic reticulum (SERCA1a), When these transmembrane sequences were displayed on a helical net, ex amination of the effects of 101 novel point mutations and 95 prior mut ations carried out on 92 transmembrane amino acids revealed ''patches' ' of sensitivity to mutation in M4, M5, and M6 but not in M8. The patc hes of mutation-sensitive residues spanned 6 of the 7 tiers of the hel ical net and covered about 240 degrees at their widest point in tiers 3 or 4 and 140 degrees in tiers 2 and 5. A contiguous column of mutati on-insensitive hydrophobic amino acids was found in M4 and M6 and in t iers 4 to 7 of M5. A six-residue motif, (E/D)GLPA(T/V) in tiers 3 and 4 of M4 and Mg with Ca2+-binding residues Glu(309) and Asp(800) as the first residue, was highlighted by mutation sensitivity, Elements of t he motif could also be discerned in M5, but reading in the C-terminal to N-terminal direction Mutation sensitivity in tier 5 of M4 mirrored mutation sensitivity of tier 5 in M6, although the amino acid sequence s were not similar, The motif or its counterpart was found in a region in M4, M5, and Mg that is made up of tiny or small amino acids but is bounded by tiers with a larger percentage of bulky amino acids, Tiers 3, 4, and 5 of M4, M5, and M6 contain Ca2+ binding and affinity mutat ions, E(1)P to E(2)P block mutations and E(2)P dephosphorylation mutat ions, indicating an important role for these central tiers in Ca2+ bin ding and in the conformational changes that accompany Ca2+ translocati on. Analysis of M8 revealed only a single mutation-sensitive residue, the Ca2+-binding amino acid, Glu(908). This residue and a mutation-ins ensitive residue, Ala(912) ,were the only vestiges of the motif that w as found in M4 and M6. Additional mutations to Glu(908) provided furth er evidence for its role in Ca2+ binding, Since mutation of M8 failed to identify residues involved in blocking conformational changes or al tering Ca2+ affinity, it is apparent that M8 plays a peripheral role i n Ca2+ binding and translocation in comparison with M4, M5, and M6.