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Biochemical and genetic investigatio...

Michigan State University.

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Biochemical and genetic investigation of the chloroplastic protein import apparatus.

Record Type:	Language materials, printed : Monograph/item
Title/Author:	Biochemical and genetic investigation of the chloroplastic protein import apparatus./
Author:	Constan, Diane Therese Jackson.
Description:	231 p.
Notes:	Adviser: Kenneth Keegstra.
Contained By:	Dissertation Abstracts International63-05B.
Subject:	Biology, Genetics. -
Online resource:	http://pqdd.sinica.edu.tw/twdaoapp/servlet/advanced?query=3053730
ISBN:	0493686924

Biochemical and genetic investigation of the chloroplastic protein import apparatus.
Constan, Diane Therese Jackson.

Biochemical and genetic investigation of the chloroplastic protein import apparatus. - 231 p.

Adviser: Kenneth Keegstra.

Thesis (Ph.D.)--Michigan State University, 2002.

Although plastids contain their own genome, almost all of the proteins needed within the organelle are encoded in the nuclear genome, synthesized on cytoplasmic ribosomes, and imported into the plastid posttranslationally. Protein import into chloroplasts is mediated by a proteinaceous complex localized within the two membranes of the plastid envelope. Several components of this translocation machinery have been identified from pea (<italic>Pisum sativum </italic>) chloroplasts, but little evidence exists concerning the individual functions of these proteins during the import process. Thus, the goal of this dissertation research has been to study the function of three import complex subunits (Tic110, Toc34, and Hsp93) in more detail. By means of experiments designed to elucidate the topology of pea Tic110 within the chloroplast inner envelope membrane, we have determined that the large C-terminal domain of Tic110, which contains the functional residues of this protein, is localized within the chloroplast stroma. Thus, Tic110 is likely involved in recruiting stromal factors, such as molecular chaperones, to the site of precursor protein translocation. The availability of the genome sequence for <italic>Arabidopsis thaliana</italic> allowed us to establish that all of the known components of the chloroplastic import apparatus are present in this species. Most of the import components have multiple homologs in <italic>Arabidopsis</italic>, suggesting that import complex composition may vary within <italic>Arabidopsis </italic> plastids. Having identified <italic>Arabidopsis</italic> homologs for the subunits of the translocation apparatus, we isolated knockout mutant lines for two putative <italic>Arabidopsis</italic> import components: AtToc34 and AtHsp93-V. Plants lacking expression of the gene encoding AtToc34 appear similar to wild-type plants, both visually and at the level of chloroplast structure and composition. In addition, <italic>in vitro</italic> import of precursor proteins is not impaired for chloroplasts isolated from the AtToc34 mutant line. Overall, we could detect no significant differences between wild-type and mutant plants. However, double mutants that lack both AtToc34 and AtToc33, a homolog of AtToc34, were not viable, indicating that AtToc33/AtToc34 function may be essential within <italic>Arabidopsis</italic> chloroplasts. A knockout mutant line for AtHsp93-V, on the other hand, is viable but much smaller and paler than wild-type plants. Chloroplasts isolated from these mutant plants contain less thylakoid membrane than do wild-type chloroplasts. These results suggest that AtHsp93-V function is necessary for normal chloroplast development. Experiments addressing whether chloroplast protein import is altered in the AtHsp93-V mutant line have given equivocal results. The rate of import of at least one precursor into isolated chloroplasts <italic>in vitro</italic> is significantly decreased in the mutant line. However, the levels of endogenous chloroplastic proteins appear to be unaffected in AtHsp93-V knockout mutant plants, suggesting that import may not be significantly impaired <italic> in vivo</italic>. This dissertation research has provided insight into the possible functions of three subunits of the chloroplastic import complex, although much still remains to be learned. It is anticipated that the tools developed during this research, especially the knockout mutant lines for AtToc34 and AtHsp93-V, will be useful for future investigations of the plastid protein import process.

ISBN: 0493686924Subjects--Topical Terms:

1017730
Biology, Genetics.

Biochemical and genetic investigation of the chloroplastic protein import apparatus.
LDR:04458nam 2200277 a 45 001 927229
005 20110425
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020 $a 0493686924
035 $a (UnM)AAI3053730
035 $a AAI3053730
040 $a UnM $c UnM
100 1 $a Constan, Diane Therese Jackson. $3 1250779
245 1 0 $a Biochemical and genetic investigation of the chloroplastic protein import apparatus.
300 $a 231 p.
500 $a Adviser: Kenneth Keegstra.
500 $a Source: Dissertation Abstracts International, Volume: 63-05, Section: B, page: 2176.
502 $a Thesis (Ph.D.)--Michigan State University, 2002.
520 $a Although plastids contain their own genome, almost all of the proteins needed within the organelle are encoded in the nuclear genome, synthesized on cytoplasmic ribosomes, and imported into the plastid posttranslationally. Protein import into chloroplasts is mediated by a proteinaceous complex localized within the two membranes of the plastid envelope. Several components of this translocation machinery have been identified from pea (<italic>Pisum sativum </italic>) chloroplasts, but little evidence exists concerning the individual functions of these proteins during the import process. Thus, the goal of this dissertation research has been to study the function of three import complex subunits (Tic110, Toc34, and Hsp93) in more detail. By means of experiments designed to elucidate the topology of pea Tic110 within the chloroplast inner envelope membrane, we have determined that the large C-terminal domain of Tic110, which contains the functional residues of this protein, is localized within the chloroplast stroma. Thus, Tic110 is likely involved in recruiting stromal factors, such as molecular chaperones, to the site of precursor protein translocation. The availability of the genome sequence for <italic>Arabidopsis thaliana</italic> allowed us to establish that all of the known components of the chloroplastic import apparatus are present in this species. Most of the import components have multiple homologs in <italic>Arabidopsis</italic>, suggesting that import complex composition may vary within <italic>Arabidopsis </italic> plastids. Having identified <italic>Arabidopsis</italic> homologs for the subunits of the translocation apparatus, we isolated knockout mutant lines for two putative <italic>Arabidopsis</italic> import components: AtToc34 and AtHsp93-V. Plants lacking expression of the gene encoding AtToc34 appear similar to wild-type plants, both visually and at the level of chloroplast structure and composition. In addition, <italic>in vitro</italic> import of precursor proteins is not impaired for chloroplasts isolated from the AtToc34 mutant line. Overall, we could detect no significant differences between wild-type and mutant plants. However, double mutants that lack both AtToc34 and AtToc33, a homolog of AtToc34, were not viable, indicating that AtToc33/AtToc34 function may be essential within <italic>Arabidopsis</italic> chloroplasts. A knockout mutant line for AtHsp93-V, on the other hand, is viable but much smaller and paler than wild-type plants. Chloroplasts isolated from these mutant plants contain less thylakoid membrane than do wild-type chloroplasts. These results suggest that AtHsp93-V function is necessary for normal chloroplast development. Experiments addressing whether chloroplast protein import is altered in the AtHsp93-V mutant line have given equivocal results. The rate of import of at least one precursor into isolated chloroplasts <italic>in vitro</italic> is significantly decreased in the mutant line. However, the levels of endogenous chloroplastic proteins appear to be unaffected in AtHsp93-V knockout mutant plants, suggesting that import may not be significantly impaired <italic> in vivo</italic>. This dissertation research has provided insight into the possible functions of three subunits of the chloroplastic import complex, although much still remains to be learned. It is anticipated that the tools developed during this research, especially the knockout mutant lines for AtToc34 and AtHsp93-V, will be useful for future investigations of the plastid protein import process.
590 $a School code: 0128.
650 4 $a Biology, Genetics. $3 1017730
650 4 $a Biology, Plant Physiology. $3 1017865
690 $a 0369
690 $a 0817
710 2 0 $a Michigan State University. $3 676168
773 0 $t Dissertation Abstracts International $g 63-05B.
790 $a 0128
790 1 0 $a Keegstra, Kenneth, $e advisor
791 $a Ph.D.
792 $a 2002
856 4 0 $u http://pqdd.sinica.edu.tw/twdaoapp/servlet/advanced?query=3053730