Chloroplast division site placement requires dimerization of the ARC11/AtMinD1 protein in Arabidopsis

Abstract

Chloroplast division is mediated by the coordinated action of a prokaryote-derived division system(s) and a host eukaryote-derived membrane fission system(s). The evolutionary conserved prokaryote-derived system comprises several nucleus-encoded proteins, two of which are thought to control division site placement at the midpoint of the organelle: a stromal ATPase MinD and a topological specificity factor MinE. Here, we show that arc11, one of 12 recessive accumulation and replication of chloroplasts (arc) mutants in Arabidopsis, contains highly elongated and multiple-arrayed chloroplasts in developing green tissues. Genomic sequence analysis revealed that arc11 contains a missense mutation in α-helix 11 of the chloroplast-targeted AtMinD1 changing an Ala at position 296 to Gly (A296G). Introduction of wild-type AtMinD1 restores the chloroplast division defects of arc11 and quantitative RT-PCR analysis showed that the degree of complementation was highly dependent on transgene expression levels. Overexpression of the mutant ARC11/AtMinD1 in transgenic plants results in the inhibition of chloroplast division, showing that the mutant protein has retained its division inhibition activity. However, in contrast to the defined and punctate intraplastidic localization patterns of an AtMinD1-YFP fusion protein, the single A296G point mutation in ARC11/AtMinD1 results in aberrant localization patterns inside chloroplasts. We further show that AtMinD1 is capable of forming homodimers and that this dimerization capacity is abolished by the A296G mutation in ARC11/AtMinD1. Our data show that arc11 is a loss-of-function mutant of AtMinD1 and suggest that the formation of functional AtMinD1 homodimers is paramount for appropriate AtMinD1 localization, ultimately ensuring correct division machinery placement and chloroplast division in plants.

The Plastid Division Protein AtMinD1 Is a Ca2+-ATPase Stimulated by AtMinE1

“…These phenotypes are reminiscent of minicelling and filamenting E. coli deficient in or overexpressing MinD (13), suggesting functional conservation between E. coli MinD and AtMinD1. In agreement with this, the polar localization of E. coli MinD reflects the distinct intraplastidic localization pattern of AtMinD1, which localizes to a single spot or two spots at opposite poles of chloroplasts (7,27).…”

“…These phenotypes are reminiscent of minicelling and filamenting E. coli deficient in or overexpressing MinD (13), suggesting functional conservation between E. coli MinD and AtMinD1. In agreement with this, the polar localization of E. coli MinD reflects the distinct intraplastidic localization pattern of AtMinD1, which localizes to a single spot or two spots at opposite poles of chloroplasts (7,27).AtMinD1 encodes a protein of 326 amino acids and, based on amino acid similarity, belongs to the ParA ATPase protein family containing a Walker A motif involved in the binding and hydrolysis of ATP. Like many ParA proteins, AtMinD1 can dimerize (27), and our studies on the accumulation and replication of chloroplasts 11 (arc11) mutant have demonstrated that the asymmetric plastid division observed in arc11 is due to a A296G missense mutation in AtMinD1 that leads to loss of dimerization and inappropriate intraplastidic localization (27).…”

“…Like many ParA proteins, AtMinD1 can dimerize (27), and our studies on the accumulation and replication of chloroplasts 11 (arc11) mutant have demonstrated that the asymmetric plastid division observed in arc11 is due to a A296G missense mutation in AtMinD1 that leads to loss of dimerization and inappropriate intraplastidic localization (27). This suggests that AtMinD1 dimerization and correct intraplastidic localization is in part important for correct Z-ring placement during Arabidopsis plastid division.…”

“…Localization-AtMinD1 shows distinct intraplastidic localization patterns localizing into one or two discrete spots at polar regions of ellipsoidal chloroplasts (7,27). In contrast, the A296G mutation in AtMinD1/ARC11 results in mislocalization, forming large and distorted fluorescent aggregates and/or multiple speckles (27).…”

“…The resulting constructs, pGADT7/AtMinD1(K72A) and pGBKT7/AtMinD1(K72A) together with pGADT7/AtMinD1 and pGBKT7/AtMinD1 (27), pGADT7/ ARC11 and pGBKT7/ARC11 (27), pGADT7/AtMinE1 and pGBKT7/AtMinE1 (29), and empty vector controls (pGADT7 and pGBKT7), were transformed into HF7c yeast cells in different combinations (Fig. 5).…”