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. 2020 Jan 22:10:1719.
doi: 10.3389/fpls.2019.01719. eCollection 2019.

Two Arabidopsis Chloroplast GrpE Homologues Exhibit Distinct Biological Activities and Can Form Homo- and Hetero-Oligomers

Pai-Hsiang Su  1   2 , Hsuan-Yu Lin  2 , Yen-Hsun Lai  2
Affiliations

Two Arabidopsis Chloroplast GrpE Homologues Exhibit Distinct Biological Activities and Can Form Homo- and Hetero-Oligomers

Pai-Hsiang Su et al. Front Plant Sci. .

Abstract

Flowering plants have evolved two distinct clades of chloroplast GrpE homologues (CGEs), which are the nucleotide exchange factor for Hsp70. In Arabidopsis, they are named AtCGE1 (At5g17710) and AtCGE2 (At1g36390). Characterization of their corresponding T-DNA insertion mutants revealed that there is no visible change in phenotype except a defect in protein import in an AtCGE2-knockout mutant under normal growth conditions. However, the embryo development of an AtCGE1-knockout mutant was arrested early at the globular stage. An AtCGE1-knockdown mutant, harboring a T-DNA insertion in the 5'-UTR region, exhibited growth retardation and protein import defect, and its mutant phenotypes became more severe when AtCGE2 was further knocked out. Sub-organellar distribution implied that AtCGE2 might be important for membrane biology due to its preferential association with chloroplast membranes. Biochemical studies and complementation tests showed that only AtCGE1, but not AtCGE2, can effectively rescue the heat-sensitive phenotype of Escherichia coli grpE mutant and robustly stimulate the refolding of denatured luciferase by DnaK. Interestingly, AtCGE1 and AtCGE2 are tending to form heterocomplexes, which exhibit comparable co-chaperone activity to AtCGE1 homocomplexes. Our data indicate that AtCGE1 is the principle functional homologue of GrpE. The possibility that AtCGE2 has a subsidiary or regulatory function through homo- and/or hetero-oligomerization is discussed.

Keywords: DnaK; Hsp70; chloroplast GrpE homologue (CGE); embryo lethal; luciferase refolding assay; oligomerization.

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Figures

Figure 1
Figure 1
A phylogenetic tree of species CGE proteins. The tree was constructed by using the neighbor-joining method in MEGA6 (19) with 62 CGE proteins (see aligned sequences in Data S1 ). Bootstrap Values are shown as a percentage of 1,000 replicates. The evolutionary distances were computed using the Dayhoff matrix based method and are in the units of the number of amino acid substitutions per site. The rate variation among sites was modeled with a gamma distribution (shape parameter = 2). All ambiguous positions were removed for each sequence pair. Al, Arabidopsis lyrata; At, Arabidopsis thaliana; Atr, Amborella trichopoda; Bd, Brachypodium distachyon; Br, Brassica rapa; Ca, Cicer arietinum; Cm, Cucumis melo; Cr, Chlamydomonas reinhardtii; Cru, Capsella rubella; Cs, Cucumis sativus; Cv, Chlorella variabilis; Es, Eutrema salsugineum; Fv, Fragaria vesca; Gm, Glycine max; Hv, Hordeum vulgare; Jc, Jatropha curcas; Mp, Micromonas pusilla; Mt, Medicago truncatula; Ob, Oryza brachyantha; OsJ, Oryza sativa Japonica Group; Ol, Ostreococcus lucimarinus; Ot, Ostreococcus tauri; Pp, Physcomitrella patens; Ppe, Prunus persica; Pt, Populus trichocarpa; Pv, Phaseolus vulgaris; Sb, Sorghum bicolor; Si, Setaria italica; Sm, Selaginella moellendorffii; St, Solanum tuberosum; Syn, Synechocystis; Ta, Triticum aestivum; Vc, Volvox carteri; Zm, Zea mays.
Figure 2
Figure 2
Characterization of atcge mutants. (A) Illustration of T-DNA insertion sites in the genomic fragments of AtCGE1 (At5g17710) and AtCGE2 (At1g36390). Both AtCGE1 and AtCGE2 contain three exons, designated as E1, E2, and E3. (B) Observing aborted seeds of atcge1-1 mutant in the siliques at the torpedo and the mature green stages under dissecting microscopy. (C) Microscopic observation of embryogenesis in wild type and atcge mutants at the torpedo stage. (D) Western blot of total chloroplast proteins isolated from wild type and atcge mutants. Samples were loaded with equal chlorophyll. Antibodies used are indicated on the right sides of blots. (E) Photograph of 21-d-old soil-grown seedlings. Fresh weight (F) and chlorophyll content (G) of the above-ground part of wild type and atcge mutants. Data are the means ± SD of three replicates.
Figure 3
Figure 3
The atcge mutants had defects in chloroplast protein import. The prRBCS import into chloroplasts isolated from wild-type and mutant seedlings grown on MS medium for 24 d. In vitro import assays were conducted for 4 to 16 min with [35S]Methionine-labeled prRBCS. After import, intact chloroplasts were reisolated and analyzed by PAGE. The gels were stained with Coomassie blue, scanned, and then dried for phosphoimaging. The region of each stained gel between the endogenous large and small subunits of Rubisco is shown below the phosphoimage. Two independent experiments (EXP1 and EXP2) are shown. TR, in vitro–translated [35S]Met-prRBCS before the import reactions.
Figure 4
Figure 4
Sub-organellar distribution of AtCGEs. Total chloroplast proteins were fractionated by ultracentrifugation after being lysed with hypotonic buffer (H.B.) or alkaline extraction (A.E.). Samples were resolved by PAGE and visualized by Western blot (WB). Coomassie Blue R-250 (CBR) stained light-harvesting chlorophyll a/b binding (LHCB) proteins and the small subunit of Rubisco (RBCS) were used as membrane and stromal protein control, respectively. Samples were loaded with equal proportion.
Figure 5
Figure 5
Functional complementation of E. coli grpE mutant (DA16) by AtCGEs. (A) Alignment of GrpE homologues. Underlines represent predicted chloroplast transit peptide (cTP), and a boxed region indicates a putative lumen transit peptide (lTP). (B) Complementation of the DA16 mutant by expressing AtCGEs. Overnight liquid culture of DA16 mutant harboring GrpE, AtCGE1, or AtCGE2 was refreshed in LB, and grown to OD600 about 0.8. A serial dilution of bacterial suspension (4 μl) was dropped on IPTG-free or -containing LB agar plates for 2 h pre-induction at 30°C, then challenged with different heat shock condition as indicated. DA15 is the wild counterpart of DA16 mutant. (C) Induction test of GrpE homologues. Arrows indicates the protein bands induced by IPTG. A dot in GrpE/IPTG-free lane indicates the leaky-expressed GrpE.
Figure 6
Figure 6
AtCGE1, but not AtCGE2, exhibited GrpE-like cochaperone activity. (A) Recombinant proteins of Dnak, DnaJ, GrpE, and AtCGEs were affinity purified, resolved on PAGE, and stained by CBR. (B) A dosage response of GrpE homologues on stimulating the luciferase refolding by DnaK/J machinery. DnaK and DnaJ concentrations are 5 and 1 μM, respectively. (C) Activity recovery of chemically denatured luciferase by DnaK/J machinery with the stimulation of wild-type or mutated AtCGE1 as indicated. The concentration of DnaK, DnaJ, and AtCGE1 are 5, 1, and 16 μM, respectively. (D) E. coli DA16 complementation by wild-type and mutated AtCGE1. After 2 h pre-induction on LB plate containing 0.05 mM IPTG at 30°C, the bacteria were challenged at 42°C for overnight.
Figure 7
Figure 7
AtCGE1/2 heterocomplex exhibited a comparable cochaperone activity with AtCGE1. (A) Affinity purification of AtCGE1/2 heterocomplex. The eluted sample contained AtCGE1 and AtCGE2-6xHis with approximately 1:1 ratio. (B) A dosage response of ACGEs on stimulating the luciferase refolding by DnaK/J machinery. DnaK and DnaJ concentrations are 5 and 1 μM, respectively. For AtCGE1/2 heterocomplex, 1 μM protein concentration equals to approximately 0.5 μM each. (C) Complementation of E. coli DA16 mutant by AtCGEs. (D) Induction of AtCGEs by IPTG. Protein bands corresponding to AtCGE1 and AtCGE2 are indicated.
Figure 8
Figure 8
Oligomeric states of AtCGEs. (A) 5 μM proteins in common buffer were crosslinked with glutaraldehyde (GA) at 0.05, 0.1, and 0.2% for 10 min. After crosslinking, their oligomeric states were visualized by 4–12% SDS-PAGE analysis and CBR staining. (B) Purified AtCGE proteins were mock incubation in common buffer, and were resolved on SDS-PAGE as uncrosslinked controls. For each lane, 1 μg protein was loaded. Asterisk (*) indicates high molecular mass oligomers.
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