Mass spectrometric genomic data mining: Novel insights into bioenergetic pathways inChlamydomonas reinhardtii

Abstract

A new high-throughput computational strategy was established that improves genomic data mining from MS experiments. The MS/MS data were analyzed by the SEQUEST search algorithm and a combination of de novo amino acid sequencing in conjunction with an error-tolerant database search tool, operating on a 256 processor computer cluster. The error-tolerant search tool, previously established as GenomicPeptideFinder (GPF), enables detection of intron-split and/or alternatively spliced peptides from MS/MS data when deduced from genomic DNA. Isolated thylakoid membranes from the eukaryotic green alga Chlamydomonas reinhardtii were separated by 1-D SDS gel electrophoresis, protein bands were excised from the gel, digested in-gel with trypsin and analyzed by coupling nano-flow LC with MS/MS. The concerted action of SEQUEST and GPF allowed identification of 2622 distinct peptides. In total 448 peptides were identified by GPF analysis alone, including 98 intron-split peptides, resulting in the identification of novel proteins, improved annotation of gene models, and evidence of alternative splicing.

Systems and Trans-System Level Analysis Identifies Conserved Iron Deficiency Responses in the Plant Lineage

“…Proteomic studies indicated that TEF22 is in the mitochondrion (Allmer et al, 2006;Atteia et al, 2009), whereas localization prediction programs (WolfPsort) place Cre14.g609900 and Cre13. g586600 at the plasma membrane.…”

“…TEF22 is induced 7.6-and 17-fold in photoheterotrophic cells in response to iron deficiency and iron limitation, respectively, and in CO 2 -grown cells even more dramatically, although the expression is always higher in acetate-grown cells. TEF22, located in the mitochondrion based on two proteomic studies (Allmer et al, 2006;Atteia et al, 2009), has a cytochrome b 561 ferric-reductase domain at its C-terminal end (see Supplemental Figure 4 online). It is distinct from the classical cytochrome b 561 -only containing proteins as TEF22 also contains a soluble heme binding DOMON domain in the central region.…”

Thylakoid FtsH Protease Contributes to Photosystem II and Cytochromeb 6 fRemodeling inChlamydomonas reinhardtiiunder Stress Conditions

“…Only two isoforms have been identified in C. reinhardtii thylakoid membranes: FtsH1 (type A) and FtsH2 (type B; Allmer et al, 2006). Our two-dimensional BN/SDS-PAGE and coimmunoprecipitation experiments demonstrate that FtsH1 also forms heterocomplexes with FtsH2 in C. reinhardtii ( Figures 3D and 3E).…”

“…Thylakoid FtsH proteases form various heterohexamers in cyanobacteria (Boehm et al, 2012) and Arabidopsis (Sakamoto et al, 2003;Yu et al, 2004;Zaltsman et al, 2005;Moldavski et al, 2012). Whereas four isoforms of thylakoid FtsH were identified in Arabidopsis (Zaltsman et al, 2005), only two thylakoid isoforms, FtsH1 and FtsH2, have been identified by proteomic studies in C. reinhardtii (Allmer et al, 2006). We raised specific antibodies against peptides from each of these isoforms (see Methods).…”

Chloroplast-mediated regulation of CO 2 -concentrating mechanism by Ca 2+ -binding protein CAS in the green alga Chlamydomonas reinhardtii

“…In Chlamydomonas, an ortholog of CAS (Cre12.g497300) was also detected in the thylakoid membrane fraction (23,24). It was proposed that CAS is required for photoacclimation by regulating induction of lightharvesting complex stress-related protein 3 (LHCSR3) (25) and for forming a super complex for photosynthetic cyclic electron flow (CEF) in anaerobic conditions (26).…”