P Champeil

Functional Properties of Sarcoplasmic Reticulum Ca2+-ATPase after Proteolytic Cleavage at Leu119-Lys120, Close to the A-domain

By measuring the phosphorylation levels of individual proteolytic fragments of SERCA1a separated by electrophoresis after their phosphorylation, we were able to study the catalytic properties of a p95C-p14N complex arising from SERCA1a cleavage by proteinase K between Leu119 and Lys120, in the loop linking the A-domain with the second transmembrane segment. ATP hydrolysis by the complex was very strongly inhibited, although ATP-dependent phosphorylation and the conversion of the ADP-sensitive E1P form to E2P still occurred at appreciable rates. However, the rate of subsequent dephosphorylation of E2P was inhibited to a dramatic extent, and this was also the case for the rate of “backdoor” formation of E2P from E2 and Pi. E2P formation from E2 at equilibrium nevertheless indicated little change in the apparent affinity for Pi or Mg2+, while binding of orthovanadate was weaker. The p95C-p14N complex also had a slightly reduced affinity for Ca2+ and exhibited a reduced rate for its Ca2+-dependent transition from E2 to Ca2E1. Thus, disruption of the N-terminal link of the A-domain with the transmembrane region seems to shift the conformational equilibria of Ca2+-ATPase from the E1/E1P toward the E2/E2P states and to increase the activation energy for dephosphorylation of Ca2+-ATPase, reviving the old idea of the A-domain being a phosphatase domain as part of the transduction machinery.

The average conformation at micromolar [Ca2+] of Ca2+-ATPase with bound nucleotide differs from that adopted with the transition state analog ADP.AlFx or with AMPPCP under crystallization conditions at millimolar [Ca2+]

Crystalline forms of detergent-solubilized sarcoplasmic reticulum Ca2+-ATPase, obtained in the presence of either a substrate analog, AMPPCP, or a transition state complex, ADP·fluoroaluminate, were recently described to share the same general architecture despite the fact that, when studied in a test tube, these forms show different functional properties. Here, we show that the differences in the properties of the E1·AMPPCP and the E1·ADP·AlFx membraneous (or solubilized) forms are much less pronounced when these properties are examined in the presence of 10 mm Ca2+ (the concentration prevailing in the crystallization media) than when they are examined in the presence of the few micromolar of Ca2+ known to be sufficient to saturate the transport sites. This concerns various properties, including ATPase susceptibility to proteolytic cleavage by proteinase K, ATPase reactivity toward SH-directed Ellmans reagent, ATPase intrinsic fluorescence properties (here described for the E1·ADP·AlFx complex for the first time), and also the rates of 45Ca2+-40Ca2+ exchange at site “II.” These results solve the above paradox at least partially and suggest that the presence of a previously unrecognized Ca2+ ion in the E1·AMPPCP crystals should be re-investigated. A contrario, they emphasize the fact that the average conformation of the E1·AMPPCP complex under usual conditions in the test tube differs from that found in the crystalline form. The extended conformation of nucleotide revealed by the E1·AMPPCP crystalline form might be only indicative of the requirements for further processing of the complex, toward the transition state leading to phosphorylation and Ca2+ occlusion.

Coordinated Overexpression in Yeast of a P4-ATPase and Its Associated Cdc50 Subunit: The Case of the Drs2p/Cdc50p Lipid Flippase Complex

Structural and functional characterization of integral membrane proteins requires milligram amounts of purified sample. Unless the protein you are studying is abundant in native membranes, it will be critical to overexpress the protein of interest in a homologous or heterologous way, and in sufficient quantities for further purification. The situation may become even more complicated if you chose to investigate the structure and function of a complex of two or more membrane proteins. Here, we describe the overexpression of a yeast lipid flippase complex, namely the P4-ATPase Drs2p and its associated subunit Cdc50p, in a coordinated manner. Moreover, we can take advantage of the fact that P4-ATPases, like most other P-type ATPases, form an acid-stable phosphorylated intermediate, to verify that the expressed complex is functional.