The Chloroplastic GrpE Homolog of Chlamydomonas: Two Isoforms Generated by Differential Splicing

Schroda, Michael; Vallon, Olivier; Whitelegge, Julian P.; Beck, Christoph F.; Wollman, Françis-André

doi:10.2307/3871537

Cited by 19 publications

(40 citation statements)

References 17 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Immunodetection was done by enhanced chemiluminescence. Antisera described previously were against HSP70B (23), CGE1 (12), mitochondrial carbonic anhydrase (33), cytochrome f (34), and CF1␤ (35).…”

Section: Methodsmentioning

confidence: 99%

“…Subsequently, lysates were loaded onto sucrose cushions (20 mM HEPES-KOH, pH 7.2, 0.6 M sucrose) and centrifuged in a Ti-50 rotor for 30 min at 152,000 ϫ g and 4°C. Antibody coupling and immunoprecipitations were carried out as described previously (12,24). Elution of immunoprecipitated proteins and cleavage of DSP were done by adding one volume of 2ϫ Laemmli sample buffer and boiling for 5 min at 95°C.…”

Section: Methodsmentioning

confidence: 99%

“…Hardly any interaction between HSP70B and CGE1 was observed in ATP-replete cell extracts. Hence, HSP70B expressed in the presence of HEP2 displayed an ATP-dependent interaction with CGE1 typical for the native protein (12,27). Because some HEP2 also was coprecipitated with CGE1, complexes composed of HSP70B, CGE1, and HEP2 must exist.…”

Section: 7 and 8)mentioning

confidence: 98%

“…Similarly important are cochaperones that regulate the affinity for nucleotides of their Hsp70 partner. For example, most prokaryotic Hsp70s require a GrpE-type nucleotide exchange factor, which is termed Mge1 in mitochondria (11) and CGE1 in chloroplasts (12).…”

mentioning

confidence: 99%

See 3 more Smart Citations

Assistance for a Chaperone

Willmund

Hinnenberger

Nick

et al. 2008

Journal of Biological Chemistry

Self Cite

View full text Add to dashboard Cite

Previous efforts aimed at the biochemical characterization of chloroplast HSP70B were hampered by the observation that recombinant HSP70B was inactive, i.e. incompetent of interacting with its nucleotide exchange factor CGE1. In addition, because heterologously expressed mitochondrial Hsp70 was inactive unless coexpressed with the escort protein Hep1, we wondered whether homologs of Hep1 existed in the chloroplast. Data base searches revealed that algae and higher plants indeed encode at least two HEP homologs, one predicted to be targeted to mitochondria, the others to chloroplasts. Using Chlamydomonas reinhardtii as plant model organism we demonstrate that this alga encodes an HEP homolog (termed HEP2) that is localized to the stroma. HEP2 is expressed constitutively as a low abundance protein with an apparent molecular mass of ϳ21 kDa. In cell extracts HEP2 interacts with HSP70B in an ATP-dependent fashion. Coexpression of HSP70B with HEP2 in Escherichia coli yielded high levels of CGE1-binding competent HSP70B, which also displayed ATPase activity. Inactive HSP70B was more prone to proteolysis than active HSP70B. Although inactive HSP70B interacted with HEP2, it could not be activated. Active HSP70B remained active for 48 h in the absence of HEP2, suggesting that HEP2 was not involved in maintaining HSP70B in an active state. However, some HSP70B expressed as a fusion protein with an N-terminal extension was activated when HEP2 was present during cleavage of the fusion protein, suggesting that in vivo HEP2 might be required for de novo folding of HSP70B after transit peptide cleavage.

show abstract

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: 7 and 8)mentioning

confidence: 98%

mentioning

confidence: 99%

See 2 more Smart Citations

Assistance for a Chaperone

Willmund

Hinnenberger

Nick

et al. 2008

Journal of Biological Chemistry

Self Cite

View full text Add to dashboard Cite

show abstract

“…37. RNA accumulations were normalized to that of the C␤LP2 transcript, which is poorly sensitive to physiological conditions (38) or 16S rRNA.…”

mentioning

confidence: 99%

Evidence for regulatory function of nucleus-encoded factors on mRNA stabilization and translation in the chloroplast

Raynaud

Loiselay

Wostrikoff

et al. 2007

Proc. Natl. Acad. Sci. U.S.A.

104

View full text Add to dashboard Cite

A salient feature of organelle gene expression is the requirement for nucleus-encoded factors that act posttranscriptionally in a gene-specific manner. A central issue is to understand whether these factors are merely constitutive or have a regulatory function. In the unicellular alga Chlamydomonas reinhardtii, expression of the chloroplast petA gene-encoding cytochrome f, a major subunit of the cytochrome b 6f complex, depends on two specific nucleusencoded factors: MCA1, required for stable accumulation of the petA transcript, and TCA1, required for its translation. We cloned the TCA1 gene, encoding a pioneer protein, and transformed appropriate mutant strains with tagged versions of MCA1 and TCA1. In transformed strains expressing decreasing amounts of MCA1 or TCA1, the concentration of these factors proved limiting for petA mRNA accumulation and cytochrome f translation, respectively. This observation suggests that in exponentially growing cells, the abundance of MCA1 sets the pool of petA transcripts, some of which are TCA1-selected for an assembly-dependent translation of cytochrome f. We show that MCA1 is a short-lived protein. Its abundance varies rapidly with physiological conditions that deeply affect expression of the petA gene in vivo, for instance in aging cultures or upon changes in nitrogen availability. We observed similar but more limited changes in the abundance of TCA1. We conclude that in conditions where de novo biogenesis of cytochrome b 6f complexes is not required, a rapid drop in MCA1 exhausts the pool of petA transcripts, and the progressive loss of TCA1 further prevents translation of cytochrome f.

show abstract

Detection and analysis of protein–protein interactions in organellar and prokaryotic proteomes by native gel electrophoresis: (Membrane) protein complexes and supercomplexes

2006

View full text Add to dashboard Cite

It is an essential and challenging task to unravel protein-protein interactions in their actual in vivo context. Native gel systems provide a separation platform allowing the analysis of protein complexes on a rather proteome-wide scale in a single experiment. This review focus on blue-native (BN)-PAGE as the most versatile and successful gel-based approach to separate soluble and membrane protein complexes of intricate protein mixtures derived from all biological sources. BN-PAGE is a charge-shift method with a running pH of 7.5 relying on the gentle binding of anionic CBB dye to all membrane and many soluble protein complexes, leading to separation of protein species essentially according to their size and superior resolution than other fractionation techniques can offer. The closely related colorless-native (CN)-PAGE, whose applicability is restricted to protein species with intrinsic negative net charge, proved to provide an especially mild separation capable of preserving weak protein-protein interactions better than BN-PAGE. The essential conditions determining the success of detecting protein-protein interactions are the sample preparations, e.g. the efficiency/mildness of the detergent solubilization of membrane protein complexes. A broad overview about the achievements of BN- and CN-PAGE studies to elucidate protein-protein interactions in organelles and prokaryotes is presented, e.g. the mitochondrial protein import machinery and oxidative phosphorylation supercomplexes. In many cases, solubilization with digitonin was demonstrated to facilitate an efficient and particularly gentle extraction of membrane protein complexes prone to dissociation by treatment with other detergents. In general, analyses of protein interactomes should be carried out by both BN- and CN-PAGE.

show abstract

The Chloroplastic GrpE Homolog of Chlamydomonas: Two Isoforms Generated by Differential Splicing

Cited by 19 publications

References 17 publications

Assistance for a Chaperone

Assistance for a Chaperone

Evidence for regulatory function of nucleus-encoded factors on mRNA stabilization and translation in the chloroplast

Detection and analysis of protein–protein interactions in organellar and prokaryotic proteomes by native gel electrophoresis: (Membrane) protein complexes and supercomplexes

Contact Info

Product

Resources

About