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

Abstract

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 NH 2 -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 ϳ25% higher affinity for its chloroplast chaperone partner HSP70B than CGE1a. Modeling of the structure of CGE1b revealed that the extended ␣-helix formed by GrpE NH 2 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 ϳ45°C). Truncating the NH 2 -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.

Assistance for a Chaperone

“…The ATP regeneration system consisted of 13.75 mM creatine phosphate and 14 g/ml creatine kinase. Immunoprecipitations were performed as described earlier (27).…”

“…Glutaraldehyde cross-links were performed as described in Ref. 27 and gel filtration analysis as reported in Ref. 25.…”

“…Be Targeted to Mitochondria and Chloroplasts-We reported recently that chloroplast HSP70B heterologously expressed in E. coli was not capable of interacting with its CGE1 cochaperone (27). Also the yeast mitochondrial Hsp70 Ssc1 was nonfunctional when it was heterologously expressed in E. coli, unless it was coexpressed with Hep1 (30).…”

“…We reported previously that Chlamydomonas HSP70B heterologously expressed in Escherichia coli was incapable of interacting with its nucleotide exchange factor CGE1; in contrast, HSP70B isolated from Chlamydomonas cell extracts did interact with CGE1 (27). These observations suggested that additional factor(s) present in its native cellular environment were required to activate HSP70B or to maintain it in an active state.…”

“…Next, pMS408 was cleaved with SapI-XhoI, and the resulting ϳ490-bp fragment was ligated into SapI-XhoI-digested pTYB11 (New England Biolabs), generating pMS410. pMS410 was transformed into strain ER2566 and, like CGE1b (pMS301) and HSP70B (pMS307), HEP2 was expressed with an intein/chitin binding domain fused to its N terminus, purified by chitin affinity chromatography, and eluted after thiol-induced cleavage as described earlier (26,27). 1 mg of purified HEP2 was used for the immunization of a rabbit.…”