Biochemistry ARTICLE Figure 1. Membrane topology analysis of MG23. (A) Signal sequence (SP) and proposed transmembrane segments in MG23 aligned sequences. Amino acid residues are numbered from the initiating methionine, and sets of identical residues among the animal species are marked with asterisks. The sites inserted with a FLAG tag for the topology analysis are also indicated. Our topology analysis predicted three transmembrane segments (TM1- TM3); the termini of each segment are tentatively assigned. Rab, rabbit; Mou, mouse; Hum, human. (B) Hydropathicity proﬁle of MG23. The hydropathicity was calculated using the Kyte-Doolittle algorithm with a window size of 19 residues. The N-terminal signal peptide (SP), putative transmembrane segments (TM1-TM3), and FLAG tag sites (Tag 1-3) are indicated. (C) Topology analysis of MG23 using skeletal muscle SR vesicles. The SR vesicles were treated with proteinase K at several concentractions (Pro K, micrograms per milliliter) in the presence or absence of 0.1% SDS. Proteolytic proﬁles were examined via Western blotting using mAb-N, mAb-C, or the antibody to calsequestrin (CSQ, SR luminal marker protein as a control). (D) Topology analysis of FLAG-tagged MG23 in ER vesicles. HEK293 cells were transfected with expression plasmids carrying FLAG- tagged MG23 cDNAs and harvested for the preparation of ER vesicles under isotonic conditions. The ER vesicles were treated with proteinase K in the presence or absence of SDS. Proteolytic proﬁles were examined via immunoblotting using mAb-N, mAb-C, mAb against the FLAG tag, or the antibody to protein disulﬁde isomerase (PDI, ER luminal marker protein). Size markers are indicated in kilodaltons. which no current was detected. Erev was corrected for the liquid junction potentials arising between the different solutions. The junction potential was calculated to be -3 mV using Clampex version 10.2 (Molecular Devices, Sunnyvale, CA). ’ RESULTS Three Transmembrane Segments Proposed for MG23. After the N-terminal signal peptide of 33 amino acid residues is removed from the primary translational product (Figure 1A), mature MG23 is localized as a 23 kDa membrane protein in the skeletal muscle SR.11 No mobility shift after the treatment of glycosidase and thiol-reducing agents in sodium dodecyl sulfate- polyacrylamide gel electrophoresis (SDS-PAGE) has suggested that mature MG23 is not subjected to obvious post-translational modifications such as glycosylation and disulfide linkage.11 Although the Prosite database search detected no conserved motif sequences or domain structures, the hydropathicity profile suggests multiple membrane-spanning segments in MG23 (Figure 1B). First, we examined the transmembrane topology of MG23 in the muscle SR. MG23 was detected as a protein band with a relative molecular mass of ∼19 kDa via SDS-PAGE. This band remained even after extensive digestion with proteinase K and was shown to be highly proteinase resistant in SR vesicles prepared under isotonic conditions. However, after proteinase treatments, we detected a cleaved product of ∼16 kDa, which was recognized by mAb-N but not by mAb-C (Figure 1C). Therefore, the proteinase-insensitive N-terminus is assigned to the luminal side, and conversely, the proteinase-sensitive C-terminus probably faces into the cytoplasm. We further introduced the FLAG tag sequence at hydrophilic sites in the MG23 primary structure and prepared ER vesicles from cultured cells expressing the FLAG-tagged MG23 proteins for proteolytic analysis (Figure 1D). The region around residue 2625 dx.doi.org/10.1021/bi1019447 |Biochemistry 2011, 50, 2623–2632