Further in vivo studies on the role of the molecular chaperone, Hsp93, in plastid protein import.
In Arabidopsis, Hsp93 is encoded by two genes, atHSP93-V and atHSP93-III. We identified two T-DNA mutants for atHSP93-III: one being a partial 'knockdown' (hsp93-III-1) and the other a complete 'knockout' (hsp93-III-2). Homozygotes for both mutants were indistinguishable from wild type. We crossed each mutant to an atHSP93-V knockout, and identified double mutants with strongly chlorotic phenotypes. This implied redundancy, which was confirmed by the complementation of mildly chlorotic hsp93-V plants by atHSP93-III over-expression. While the hsp93-V hsp93-III-1 mutant was doubly homozygous, the second double mutant was heterozygous for hsp93-III-2 (genotype: hsp93-V/hsp93-V; +/hsp93-III-2). Attempts to identify an hsp93-V hsp93-III-2 double homozygote were unsuccessful, indicating that the Hsp93 pool is essential for viability. Consistently, siliques of the second double mutant contained aborted seeds (because of a block in the zygote-embryo transition) and failed ovules (because of a moderate defect in female gametophytes). Double-mutant plants were chlorophyll-deficient, contained under-developed chloroplasts, and exhibited stunted growth. In import assays using a chimeric pre-protein (plastocyanin transit peptide fused to dihydrofolate reductase; PC-DHFR), a clear defect was observed in hsp93-V hsp93-III-1 chloroplasts. Interestingly, while denaturation or stabilization of the DHFR moiety had a strong effect on import efficiency in the wild type, no such effects were observed with double-mutant (or tic40) chloroplasts. This indicated that pre-protein unfolding is not rate-limiting for import into mutant chloroplasts, and suggested that (unlike the situation in mitochondria) the inner membrane import machinery does not contribute to pre-protein unfolding at the organellar surface.
Kovacheva
Sabina
S
Department of Biology, University of Leicester, Leicester LE1 7RH, UK.
Bédard
Jocelyn
J
Wardle
Anthony
A
Patel
Ramesh
R
Jarvis
Paul
P
D000818:Animals; D029681:Arabidopsis Proteins / Q000235:genetics / Q000502:physiology*; D002734:Chlorophyll / Q000378:metabolism; D002736:Chloroplasts / Q000378:metabolism / Q000648:ultrastructure; D006360:Heat-Shock Proteins / Q000235:genetics / Q000502:physiology*; D015151:Immunoblotting; D051379:Mice; D046529:Microscopy, Electron, Transmission; D018832:Molecular Chaperones / Q000235:genetics / Q000502:physiology*; D009154:Mutation; D010641:Phenotype; D030821:Plants, Genetically Modified; D018087:Plastids / Q000378:metabolism*; D021381:Protein Transport / Q000235:genetics / Q000502:physiology; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012639:Seeds / Q000166:cytology / Q000235:genetics / Q000378:metabolism; D013762:Tetrahydrofolate Dehydrogenase / Q000235:genetics / Q000378:metabolism
D029681:Arabidopsis Proteins; D006360:Heat-Shock Proteins; D018832:Molecular Chaperones; D011993:Recombinant Fusion Proteins; D002734:Chlorophyll; D013762:Tetrahydrofolate Dehydrogenase