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CPSRP54 and co-translational targeting of chloroplast encoded thylakoid membrane proteins
Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
2003 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Chloroplasts are essential organelles of prokaryotic origin that are present in green tissue of higher plants and algae. Chloroplasts are predicted to contain ~3000 proteins and the vast majority of these proteins are encoded by nuclear genes. Chloroplasts also have a small circular genome encoding 100-120 tRNAs, rRNAs and proteins. About 30 of these chloroplast encoded proteins are integral thylakoid membrane proteins. It is very important to understand how these membrane proteins are targeted, inserted and assembled, since most are essential core components of the four thylakoid membrane protein complexes (PSI, PSII, cytf and ATP synthase) that convert light energy into chemical energy. Chloroplasts have inherited protein targeting and insertion pathways from its prokaryotic progenitor. One of these pathways is the Signal Recognition Particle (SRP) dependent pathway, which evolved from a strict co-translational pathway in bacteria, into a pathway that also functions in post-translational targeting of nuclear encoded thylakoid membrane proteins. This thesis concerns the role of the chloroplast localized 54 kDa subunit of SRP (cpSRP54) in targeting and insertion of the chloroplast-encoded thylakoid membrane proteins. In vitro reconstitution studies and in vivo analysis of wild-type and a cpSRP54 Arabidopsis thaliana null mutant were combined to address molecular details of SRP - substrate interaction and to follow the fate of cpSRP dependent substrates, as well as possible compensation mechanisms. A homologous in vitro chloroplast translation system was used to investigate the co-translational targeting function of cpSRP54. Amber suppression was adapted to the homologous translation system and was used to incorporate photo-activated crosslinkers at site-specific positions in D1 nascent chains. Together with conventional chemical crosslinking techniques, this was used to demonstrate that cpSRP54 transiently interacts with elongating nascent chains of D1, a chloroplast-encoded thylakoid membrane protein. CpSRP54 interaction was shown to depend on the hydrophobicity of the first transmembrane domain when it emerged from the ribosome. CpSRP54 was not able to interact with free nascent chains and no direct interaction between nascent chains and cpSecA or cpSRP43 could be found. This supports a conserved function of cpSRP54 in targeting of chloroplast encoded thylakoid membrane proteins. A detailed study of a cpSRP54 A. thaliana disruption mutant was carried out to assess cpSRP54 function in vivo and to follow accumulation of (potential) cpSRP substrates and components of the targeting machinery during leaf development. Plants lacking cpSRP54 were delayed in both growth and development at all light regimes tested. The translocon component CpSecY, involved in translocation of a subset of lumenal proteins and possibly also involved in co-translational membrane insertion of the D1 protein, remained at a constant level throughout leaf expansion and maturation. In contrast, the thylakoid membrane translocon ALB3, as well as other targeting components (cpSRP43, cpSRP54 and cpFtsY) were down regulated with progressive leaf development. Importantly, the down-regulation of ALB3 was strongly delayed in the cpSRP54 mutant, whereas no clear difference could be seen in accumulation of cpSRP43, cpFtsY and cpSecY between wt and the cpSRP54 mutant. Accumulation of nuclear encoded LHCIIs, post-translationally targeted by cpSRP54 and three chloroplast-encoded PSII core subunits was also delayed in the cpSRP54 mutant. It is concluded that cpSRP54 function in post- and co-translational protein targeting of proteins to the thylakoid membrane in chloroplasts.

Place, publisher, year, edition, pages
Stockholm, 2003. , 52 p.
National Category
Medical Biotechnology (with a focus on Cell Biology (including Stem Cell Biology), Molecular Biology, Microbiology, Biochemistry or Biopharmacy)
Research subject
URN: urn:nbn:se:su:diva-28801ISBN: 91-7265-667-0OAI: diva2:226508
Public defence
2003-05-22, Magnélisalen, Kemiska övningslaboratoriet, Frescati, Svante Arrhenius väg 12 A, Stockholm, 10:00 (English)
Därtill 4 uppsatserAvailable from: 2009-06-30 Created: 2009-06-30 Last updated: 2009-12-15Bibliographically approved

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Medical Biotechnology (with a focus on Cell Biology (including Stem Cell Biology), Molecular Biology, Microbiology, Biochemistry or Biopharmacy)

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