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J. Biol. Chem., Vol. 282, Issue 15, 11317-11328, April 13, 2007

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The NH₂-terminal Domain of the Chloroplast GrpE Homolog CGE1 Is Required for Dimerization and Cochaperone Function in Vivo^*

From the Institute of Biology II, Plant Biochemistry at the University of Freiburg, Freiburg D-79104, Germany

GrpE proteins function as nucleotide exchange factors for DnaK-type Hsp70s. We have previously identified a chloroplast homolog of GrpE in Chlamydomonas reinhardtii, termed CGE1. CGE1 exists as two isoforms, CGE1a and CGE1b, which are generated by temperature-dependent alternative splicing. CGE1b contains additional valine and glutamine residues in its extreme NH₂-terminal region. Here we show that CGE1a is predominant at lower temperatures but that CGE1b becomes as abundant as CGE1a at elevated temperatures. Coimmunoprecipitation experiments revealed that CGE1b had a 25% higher affinity for its chloroplast chaperone partner HSP70B than CGE1a. Modeling of the structure of CGE1b revealed that the extended -helix formed by GrpE NH₂ termini is 34 amino acids longer in CGE1 than in Escherichia coli GrpE and appears to contain a coiled coil motif. Progressive deletions of this coiled coil increasingly impaired the ability of CGE1 to form dimers, to interact with DnaK at elevated temperatures, and to complement temperature-sensitive growth of a grpE E. coli strain. In contrast, deletion of the four-helix bundle required for dimerization of E. coli GrpE did not affect CGE1 dimer formation. Circular dichroism measurements revealed that CGE1, like GrpE, undergoes two thermal transitions, the first of which is in the physiologically relevant temperature range (midpoint 45 °C). Truncating the NH₂-terminal coiled coil shifted the second transition to lower temperatures, whereas removal of the four-helix bundle abolished the first transition. Our data suggest that bacterial GrpE and chloroplast CGE1 share similar structural and biochemical properties, but some of these, like dimerization, are realized by different domains.

Received for publication, September 14, 2006 , and in revised form, February 8, 2007.

^* This work was supported by Deutsche Forschungsgemeinschaft Grants Schr 617/2-2 and 617/2-3. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ To whom correspondence should be addressed: Institute of Biology II, Schänzlestr. 1, D-79104 Freiburg, Germany. Tel.: 49-761/203-2708; Fax: 49-761/203-2601; E-mail: michael.schroda{at}biologie.uni-freiburg.de.

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This article has been cited by other articles:

				F. Willmund, M. Hinnenberger, S. Nick, M. Schulz-Raffelt, T. Muhlhaus, and M. Schroda Assistance for a Chaperone: CHLAMYDOMONAS HEP2 ACTIVATES PLASTIDIC HSP70B FOR COCHAPERONE BINDING J. Biol. Chem., June 13, 2008; 283(24): 16363 - 16373. [Abstract] [Full Text] [PDF]