Biochemistry Figure 2. Puriﬁcation and cross-linking of MG23. (A) Puriﬁcation of recombinant MG23 from cDNA-transfected yeast cells. The cell lysate fraction, microsomal fraction, Ni aﬃnity-puriﬁed fraction, and immu- noaﬃnity-puriﬁed fraction were analyzed via SDS-PAGE. Proteins were visualized by Coomassie blue staining, and recombinant MG23 is marked with an arrowhead. (B) Cross-linking of puriﬁed recombinant MG23. An aﬃnity-puriﬁed recombinant MG23 preparation was treated with the cross-linker disuccinimidyl glutarate at concentrations of 10-6-10-3 M, and the resulting products were examined by immuno- blotting using mAb-N; monomeric to hexameric products are num- bered. Size markers are indicated in kilodaltons. 119 (Tag 2 site) was resistant to proteinase digestion but became proteinase-sensitive when the FLAG tag was introduced. Therefore, the Tag 2 site is reasonably assigned to the cyto- plasmic side and may be associated with the membrane environment in the original form of mature MG23. On the other hand, no obvious diﬀerence in digestion pattern was observed by insertion of the tag at the Tag 1 and Tag 3 sites, suggesting their localization on the luminal side of the ER/SR. Taken together, the proteolytic analysis data predict three transmembrane segments, which are arranged in MG23 ac- cording to the positive-inside rule.29 In this model (Figure 3C), of the 210 amino acid residues that composed the mature MG23, the cytoplasmic region contains 59 residues (28%), the transmembrane region carries 65 residues (31%), and the luminal region possesses 86 residues (41%). Homo-Oligomeric Assembly of MG23. Native MG23 was solubilized from rabbit SR vesicles and purified using mAb affinity chromatography (Figure S1A of the Supporting In- formation). Although purified preparations contained a few impurities, including Ca2þ-ATPase (∼100 kDa protein), no proteins were coenriched with MG23 during the affinity pur- ification. Moreover, recombinant His-tagged MG23 was ex- pressed in metanotrophic yeast (Figure 2A) and highly purified using combined affinity chromatography (>95% purity via SDS- PAGE). Both preparations were used to characterize the struc- tural and electrophysiological properties of MG23 in this study. When native and recombinant MG23 preparations were reacted with chemical cross-linkers, immunoblotting detected the gen- eration of homo-oligomeric products (Figure 2B). For example, cross-linking by disuccinimidyl glutarate generated oligomeric complexes, up to a hexamer, in both native and recombinant MG23 preparations (Figure S1B of the Supporting Information). The observations suggest that MG23 forms at least a homohex- amer as a putative self-assembly unit. ARTICLE MG23 Particle Imaging. After the negative staining, purified recombinant MG23 was examined by EM (Figure 3A). Most particles were round-shaped or bowl-shaped, which probably represent vertical or lateral views, respectively. We frequently detected round-shaped particles with electron-dense rim, inter- preted to be the views of the MG23 particle perpendicular to the membrane. Putative side views clearly show asymmetry with respect to the membrane; one end is wider and the other narrower. To examine the topological orientation to the SR/ER membrane, recombinant MG23 particles were reacted with mAb to the N-terminal His tag (mAb-His) and mAb-C, and the resulting MG23-mAb complexes were analyzed by EM (Figure 3B). Although we frequently detected MG23 particles bearing multiple antibodies, mAb-His predominantly attached to the wider side of the particle, while mAb-C reacted with the narrower side. The EM observation, together with the proteolytic topology assay, suggests that the narrow side of the MG23 particle faces the cytoplasm while the wide side is oriented to the ER/SR lumen (Figure 3C). 3D Reconstruction of Bowl-Shaped MG23 Particles. To understand the molecular shape of MG23, we reconstructed its 3D structure by the single-particle reconstruction technique. Purified recombinant MG23 protein was adsorbed to the grid, blotted with the filter paper, negatively stained, and recorded by EM. MG23 particles were selected from the digitized films using our program, and a total of 11175 images were picked up for 3D reconstruction (Figure 4). The surface representation demon- strates that MG23 is a bowl-shaped molecule, 12 nm in height, 16 nm in side length, and 17 nm diagonally at the widest transmembrane region. Because of the symmetry of the averaged top view (Figure 4A, left-most panel, second row), we applied 6-fold symmetry in this reconstruction. Projection from the final density map (Figure 4A, row 4) compares well with the corres- ponding raw particle images (Figure 4A, row 1), indicating that our 3D reconstruction is consistent with the data set. The presumed position of the membrane is indicated by the trans- parent blue band in Figure 3C, so that the volume ratio across the membrane fits to the topology model based on the hydropathi- city and protein chemical data. In height, the transmembrane region of the bowl-shaped structure is ∼3 nm, the cytoplasmic domain extends ∼5 nm, and the luminal domain extends ∼4 nm. In the serial sections of this structure (Figure S2 of the Support- ing Information), the thickness of the lateral wall is 28-35 Å in the transmembrane region. A plot of the Euler angles of 163 adopted class averages shows the nearly random orientation of MG23 on the grid surface (Figure S3A of the Supporting Information). According to the Fourier shell correlation (FSC) function, the resolution limit for the reconstruction is 2.0 nm by the correlation coefficient of 0.5 criterion (Figure S3B of the Supporting Information). The final reconstruction included 9172 particle images (82% of all the selected images). 3D Reconstruction of Smaller-Sized MG23 Particles. Dur- ing the EM analysis described above, we realized that the recombinant MG23 preparations always contained considerable amounts of asymmetric particles that were relatively smaller than the bowl-shaped particle (Figure 3A). We also detected similar EM images showing the coexistence of the larger and smaller particles in native MG23 preparations purified from rabbit skeletal muscle (data not shown). Therefore, we also tried to reconstruct the 3D structure of the smaller-sized particles. A total of 1707 images of the smaller particles were collected from the digitized films and used for 3D reconstruction. Because no symmetry was observed in the averaged images or the raw 2626 dx.doi.org/10.1021/bi1019447 |Biochemistry 2011, 50, 2623–2632