A novel membrane protein in spinich chloroplast and its homologue in Arabidopsis thaliana

Abstract

Two-dimensional gel electrophoresis (2-D) of proteins combining with microsequencing analysis provides a powerful approach for the identification of thylakoid membrane proteins and the N-terminal sequence information for cloning of corresponding genes [see Shi-Gui Yu et al., Photosyn. Res. 41(1994) 475-486]. In order to purify and to characterize protein spots in the basic range of 2-D, we have modified the non-equilibrium pH-gradient electrophoresis (NEPHGE) gel system and adapted it to the membrane protein analysis. The 2-D protein pattern of spinach thylakoid membranes from the NEPHGE analysis shows about 50 polypeptide spots in the basic Ip area. On the basis of microsequence data and sequence comparison through databases, it has been found that a protein having a molecular size of 40 kDa and focusing at pH range of 7.8 is a novel one and its N-terminal 18 amino acid sequence is VSLPKEQLVTSLTQVEQT. This newly discovered protein has 66.7% identity and 100% similarity with the N-terminal region of a protein sequence deduced from EST (Expressed Sequence Tag) and the DNA sequence of the chromosome 5 from Arabidopsis thaliana. The molecular mass estimated from the amino acid sequence of this nuclei-encoded protein is also about 40 kDa which fits well with the novel one. The secondary structure analysis of this protein demonstrates a typical membrane spanning domain in its N-terminal region. Protein analysis for thylakoid fragments derived from different parts of the spinach thylakoids reveals that the novel protein is localized in both the stroma and the grana lamellae of chloroplasts.