Crystallization of a mammalian membrane protein overexpressed in Saccharomyces cerevisiae

Abstract

The Ca 2؉ -ATPase SERCA1a (sarcoplasmic-endoplasmic reticulum Ca 2؉ -ATPase isoform 1a) from rabbit has been overexpressed in Saccharomyces cerevisiae. This membrane protein was purified by avidin agarose affinity chromatography based on natural biotinylation in the expression host, followed by HPLC gel filtration. Both the functional and structural properties of the overexpressed protein validate the method. Thus, calcium-dependent ATPase activity and calcium transport are essentially intact after reconstitution in proteoliposomes. Moreover, the recombinant protein crystallizes in a form that is isomorphous to the native SERCA1a protein from rabbit, and the diffraction properties are similar. This represents a successful crystallization of a mammalian membrane protein derived from a heterologous expression system, and it opens the way for the study of mutant forms of SERCA1a.overexpression ͉ Ca 2ϩ -ATPase ͉ SERCA1a T he major protein component of the sarcoplasmic reticulum (SR) membrane is a Ca 2ϩ -transporting ATPase that is responsible for the cessation of muscle contraction cycle. Since its discovery (1), rabbit SR Ca 2ϩ -ATPase (SERCA1a, sarcoplasmic-endoplasmic reticulum Ca 2ϩ -ATPase isoform 1a) has been a favorite subject for membrane studies, because vesicles predominantly consisting of Ca 2ϩ -ATPase and with intact Ca 2ϩ -transporting properties can be prepared in large yield from skeletal muscle. Moreover, this protein is a paradigm of P-type ATPases, which constitute the major cation transporter family. P-type ATPases are characterized by the formation of a covalent aspartylphosphorylated intermediate during their catalytic cycle. Crystallization attempts of SERCA1a started a long time ago from detergent-solubilized membranes, which provided valuable initial structural information as to how to proceed with crystallization of this difficult-to-handle membrane protein (2, 3). Recently, several 3D structures corresponding to various conformations of the catalytic cycle have been described, all of them obtained from the native protein of SR (4-10). With the object of studying structural and functional properties of mutants of this transporter, we here outline a procedure of purification of Ca 2ϩ -ATPase after heterologous expression in yeast (11). Our aim was to increase the quantity and, most importantly, the quality of our purified protein to be used for 3D crystallization. For this, we have chosen to use a recombinant protein: the SERCA1a fused to a biotin acceptor domain (SERCA1a-BAD). This procedure allows us to specifically purify the fusion protein by affinity chromatography after in vivo biotinylation, which selects for properly folded protein. Finally, we show, with wild-type SERCA1a as an example, that the purified protein can be used for crystallization and functional studies, including transport, and that its structure is isomorphous to the native enzyme from rabbit. MethodsProtein Purification. Expression in yeast was performed as described in ref. 11. Starting from a 4-liter culture, 75 ml ...

Manipulating phospholipids for crystallization of a membrane transport protein

“…However, it has also been reported recently (7) that crystals of cytochrome b 6 f that diffract well can be obtained only by adding PL, which stabilize the intact complex. Successful crystallization of a heterologously expressed rabbit sarcoplasmic-endoplasmic reticulum Ca 2ϩ -ATPase isoform also requires addition of native PL (8). In addition, the amount of residual PL associated with the P i ͞glycerol-P antiporter GlpT is important for crystallization (9).…”

GFP-based optimization scheme for the overexpression and purification of eukaryotic membrane proteins in Saccharomyces cerevisiae

“…Although more structures of eukaryotic membrane proteins have been determined using material isolated from heterologous overexpression in the methylotrophic yeast Pichia pastoris, this difference is likely due to the popularity of P. pastoris because of its ability to produce a large biomass during fermentation with methanol 8 . However, recent work has demonstrated that S. cerevisiae is also a suitable host for obtaining enough material that will give high-resolution membrane protein structures 9,10 .…”

Crystallization and preliminary structural analysis of theListeria monocytogenesCa2+-ATPase LMCA1

“…3). This type I packing is also observed for re-lipidated SERCA1a (Jidenko et al, 2005) and in general for ATPases solubilized from native tissue: SERCA1a (Toyoshima et al, 2000;Sørensen et al, 2004) and Na + ,K + -ATPase . The molecular-replacement solution was subjected to rigid-body and limited restrained refinement (R = 44.1% and R free = 47.4%).…”

“…LMCA1 eluted as a monomer in size-exclusion chromatography, similar to C 12 E 8 -solubilized SERCA1a from rabbit SR membranes (Andersen et al, 1986). Prior to crystallization, LMCA1 was re-lipidated with 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) as previously described for SERCA1a expressed in yeast (Jidenko et al, 2005). Excess lipid that was not solubilized by the detergent was removed by ultracentrigufation and LMCA1 was incubated with the Ca 2+ chelator EGTA and AlF 4 À to mimic the E2-P i -occluded form as a transition state in dephosphorylation.…”

“…A weight ratio of 1:3 DOPC:C 12 E 8 was achieved by adding LMCA1 to a glass tube pre-treated with a thin DOPC film. The weight ratio was calculated assuming all-micellar C 12 E 8 being concentrated with LMCA1 in the ultrafiltration step (Jidenko et al, 2005). The lipid film was generated by dispensing DOPC dissolved in CHCl 3 into the glass tube and evaporating the CHCl 3 in an N 2 atmosphere, thus preventing oxidation.…”