The regulation and function of chimaerins, a family of "non-protein kinase C" (PKC) phorbol ester/diacylglycerol receptors with Rac-GAP activity, is largely unknown. In a search for chimaerin-interacting proteins, we isolated Tmp21-I (p23), a protein localized at the perinuclear Golgi area. Remarkably, phorbol esters translocate 2-chimaerin to the perinuclear region and promote its association with Tmp21-I in a PKC-independent manner. A deletional analysis revealed that the C1 domain in chimaerins is required for the interaction with Tmp21-I, thereby implying a novel function for this domain in protein-protein associations in addition to its role in lipid and phorbol ester binding. Our results support the emerging concept that multiple pathways transduce signaling by phorbol esters and revealed that, like PKC isozymes, chimaerins are subject to a positional regulation. In this setting, Tmp21-I serves as an anchoring protein that determines the intracellular localization of these novel phorbol ester receptors. Protein kinase C (PKC)1 isozymes, a family of related serine/ threonine kinases, were the first receptors isolated for the phorbol ester tumor promoters and the second messenger diacylglycerol (DAG) (1, 2). It is now well established that phorbol esters and DAG also bind to multiple proteins lacking kinase activity, including the Rac GTPase-activating proteins (GAPs) ␣-and -chimaerins, the Ras exchange factor RasGRP, and the Unc-13/Munc13 family of scaffolding proteins. These novel "non-kinase" phorbol ester receptors possess a single copy of the C1 domain, the cysteine-rich motif responsible for binding of phorbol esters and DAG. Two copies of the motif (C1a and C1b) are present in the regulatory domain of phorbol esterresponsive PKCs (classical PKCs, cPKCs, and novel PKCs, nPKCs). The 50 or 51 amino acid C1 domains have the motif HX 12 CX 2 CX 13/14 CX 2 CX 4 HX 2 CX 7 C, where X is any other amino acid (3-5). C1 domains are required for the association of PKCs and novel non-kinase phorbol ester receptors with membranes (6 -8).The chimaerin family of phorbol ester receptors includes four related isoforms (␣1-or n-, ␣2-, 1-, and 2-chimaerin), which are spliced variants from the ␣-and -chimaerin genes. The C1 domain in chimaerin isoforms has ϳ40% identity with those in PKC isozymes. The identity between the C1 domains of ␣-and -chimaerins is 94% (3, 9 -11). The C-terminal region of chimaerins possesses high homology to BCR, the breakpoint cluster region protein involved in Philadelphia chromosome translocation in chronic myelogenous leukemia. This domain was reported to have GAP activity for Rac (12), a small GTP-binding protein that plays a key role in actin cytoskeleton organization, adhesion, migration, gene expression, and mitogenesis (13). The main structural difference among chimaerin isoforms is the presence of a putative N-terminal SH2 domain in the spliced forms ␣2-and 2-chimaerin. Although the distribution of ␣1-and 1-chimaerin is restricted mainly to brain and testis, respectively, ␣2-and...
Calcium is important for chloroplast, not only in its photosynthetic but also nonphotosynthetic functions. Multiple Ca(2+)/H(+) transporters and channels have been described and studied in the plasma membrane and organelle membranes of plant cells; however, the molecular identity and physiological roles of chloroplast Ca(2+)/H(+) antiporters have remained unknown. Here we report the identification and characterization of a member of the UPF0016 family, CCHA1 (a chloroplast-localized potential Ca(2+)/H(+) antiporter), in Arabidopsis thaliana. We observed that the ccha1 mutant plants developed pale green leaves and showed severely stunted growth along with impaired photosystem II (PSII) function. CCHA1 localizes to the chloroplasts, and the levels of the PSII core subunits and the oxygen-evolving complex were significantly decreased in the ccha1 mutants compared with the wild type. In high Ca(2+) concentrations, Arabidopsis CCHA1 partially rescued the growth defect of yeast gdt1Δ null mutant, which is defective in a Ca(2+)/H(+) antiporter. The ccha1 mutant plants also showed significant sensitivity to high concentrations of CaCl2 and MnCl2, as well as variation in pH. Taken these results together, we propose that CCHA1 might encode a putative chloroplast-localized Ca(2+)/H(+) antiporter with critical functions in the regulation of PSII and in chloroplast Ca(2+) and pH homeostasis in Arabidopsis.
In chloroplasts, thioredoxin (TRX) isoforms and NADPH-dependent thioredoxin reductase C (NTRC) act as redox regulatory factors involved in multiple plastid biogenesis and metabolic processes. To date, less is known about the functional coordination between TRXs and NTRC in chlorophyll biosynthesis. In this study, we aimed to explore the potential functions of TRX m and NTRC in the regulation of the tetrapyrrole biosynthesis (TBS) pathway. Silencing of three genes, TRX m1, TRX m2, and TRX m4 (TRX ms), led to pale-green leaves, a significantly reduced 5-aminolevulinic acid (ALA)-synthesizing capacity, and reduced accumulation of chlorophyll and its metabolic intermediates in Arabidopsis (Arabidopsis thaliana). The contents of ALA dehydratase, protoporphyrinogen IX oxidase, the I subunit of Mg-chelatase, Mg-protoporphyrin IX methyltransferase (CHLM), and NADPH-protochlorophyllide oxidoreductase were decreased in triple TRX m-silenced seedlings compared with the wild type, although the transcript levels of the corresponding genes were not altered significantly. Protein-protein interaction analyses revealed a physical interaction between the TRX m isoforms and CHLM. 4-Acetoamido-4-maleimidylstilbene-2,2-disulfonate labeling showed the regulatory impact of TRX ms on the CHLM redox status. Since CHLM also is regulated by NTRC , we assessed the concurrent functions of TRX m and NTRC in the control of CHLM. Combined deficiencies of three TRX m isoforms and NTRC led to a cumulative decrease in leaf pigmentation, TBS intermediate contents, ALA synthesis rate, and CHLM activity. We discuss the coordinated roles of TRX m and NTRC in the redox control of CHLM stability with its corollary activity in the TBS pathway.
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