Figure 5.

Phylogenic Tree and Topology Analysis of the GOT1B Protein.

(A) Phylogenic tree of the GOT1B protein and its homologs in eukaryotes. The phylogenic tree was constructed using MEGA version 5.0. Sb, Sorghum bicolor; Zm, Zea mays; Hv, Hordeum vulgare; Os, Oryza sativa; Si, Setaria italica; At, Arabidopsis thaliana; Br, Brassica rapa; Gs, Glycine soja; Mt, Medicago truncatula; H, Homo sapiens; Dm, Drosophila melanogaster; Mm, Mus musculus; Pt, Pan troglodytes; Oa, Ovis aries; Ss, Sus scrofa. The sequence alignment used for this analysis is available as Supplemental Data Set 1.

(B) Sequence alignment of GOT1B and its homologous proteins. Red lines indicate the four transmembrane domains predicted by TMHMM Server v. 2.0 (http://www.cbs.dtu.dk/services/TMHMM).

(C) Membrane association of GOT1B protein expressed in N. benthamiana. GOT1B was expressed in tobacco leaf epidermal cells. The 100,000g membrane pellets were prepared and extracted with indicated buffers before centrifugation and analysis of the pellets (P100) and supernatants (S100) by immunoblot. The closed arrowhead indicates the GOT1B protein band. The open arrowhead indicates the Tip3-1 protein band.

(D) and (E) Protease protection assays. Membrane preparations containing the GOT1B protein tagged at either the C (D) or N terminus (E), as indicated, were digested with proteinase K in the presence or absence of detergent and then analyzed by immunoblot.

(F) The proposed topology of GOT1B protein. TMD, transmembrane domain.