Identification and functional analysis of thylakoid membrane proteome

Wang, Yingchun
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Membrane proteins play crucial roles in many biological functions. Identities and functions of most membrane proteins remain to be revealed. New technological breakthroughs in proteomics together with the availability of genomic sequence information make it possible to study functions of membrane proteins on a genome-wide scale. We used a multidisciplinary approach combining biochemistry, genetics, proteomics and bioinformatics to study the functions of the thylakoid proteome of Synechocystis sp. PCC6803. The thylakoid membrane proteins were separated into peripheral and integral fractions and resolved into 2-D gels with different pH ranges. The protein spots in the 2-D gels were subjected to peptide mass fingerprinting analysis, and totally 390 out of 558 analyzed spots were identified as protein products of 128 individual genes, of which 38 gene encode hypothetical proteins with unknown function. To study the function of some hypothetical proteins, we inactivated a set of genes, and 10 knockout mutants were obtained. The growth analysis for the mutant cells revealed that only one mutant (H1) which has a deletion in the ORF slr0110, showed conditional growth phenotype. Detailed analysis indicated that the H1 mutant is sensitive to both glucose and light, which is caused by the over-reduction of the PQ pool in the thylakoid membrane. The ID and the structural and functional information of the identified proteins as well as the 2-D reference maps were included in a web-based relational database for thylakoid membrane proteins. The database was constructed with MySQL, and the application programs were developed with SQL, PERL, JAVASCRIPT and HTML. Users can search the information of identified proteins and compare their own identified proteins with the identified proteins in the database. A manager interface is also provided for the routine maintenance of the database.

Biochemistry, biophysics, and molecular biology, Electrical and computer engineering, Genetics, Bioinformatics and computational biology