Discovery of a Unique Clp Component, ClpF, in Chloroplasts: A Proposed Binary ClpF-ClpS1 Adaptor Complex Functions in Substrate Recognition and Delivery.

Clp proteases are found in prokaryotes, mitochondria, and plastids where they play crucial roles in maintaining protein homeostasis (proteostasis). The plant plastid Clp machinery comprises a hetero-oligomeric ClpPRT proteolytic core, ATP-dependent chaperones ClpC and ClpD, and an adaptor protein, ClpS1. ClpS1 selects substrates to the ClpPR protease-ClpC chaperone complex for degradation, but the underlying substrate recognition and delivery mechanisms are currently unclear. Here, we characterize a ClpS1-interacting protein in Arabidopsis thaliana, ClpF, which can interact with the Clp substrate glutamyl-tRNA reductase. ClpF and ClpS1 mutually stimulate their association with ClpC. ClpF, which is only found in photosynthetic eukaryotes, contains bacterial uvrB/C and YccV protein domains and a unique N-terminal domain. We propose a testable model in which ClpS1 and ClpF form a binary adaptor for selective substrate recognition and delivery to ClpC, reflecting an evolutionary adaptation of the Clp system to the plastid proteome.

Posttranslational Control of ALA Synthesis Includes GluTR Degradation by Clp Protease and Stabilization by GluTR-Binding Protein.

5-Aminolevulinic acid (ALA) is the first committed substrate of tetrapyrrole biosynthesis and is formed from glutamyl-tRNA by two enzymatic steps. Glutamyl-tRNA reductase (GluTR) as the first enzyme of ALA synthesis is encoded by HEMA genes and tightly regulated at the transcriptional and posttranslational levels. Here, we show that the caseinolytic protease (Clp) substrate adaptor ClpS1 and the ClpC1 chaperone as well as the GluTR-binding protein (GBP) interact with the N terminus of GluTR Loss-of function mutants of ClpR2 and ClpC1 proteins show increased GluTR stability, whereas absence of GBP results in decreased GluTR stability. Thus, the Clp protease system and GBP contribute to GluTR accumulation levels, and thereby the rate-limiting ALA synthesis. These findings are supported with Arabidopsis (Arabidopsis thaliana) hema1 mutants expressing a truncated GluTR lacking the 29 N-terminal amino acid residues of the mature protein. Accumulation of this truncated GluTR is higher in dark periods, resulting in increased protochlorophyllide content. It is proposed that the proteolytic activity of Clp protease counteracts GBP binding to assure the appropriate content of GluTR and the adequate ALA synthesis for chlorophyll and heme in higher plants.

GluTR2 complements a hema1 mutant lacking glutamyl-tRNA reductase 1, but is differently regulated at the post-translational level.

Arabidopsis HEMA1 and HEMA2 encode glutamyl-tRNA reductase (GluTR) 1 and 2, the two isoforms of the initial enzyme of tetrapyrrole biosynthesis. HEMA1 is dominantly expressed in photosynthetic tissue, while HEMA2 shows low constitutive expression and is induced upon stress treatments. We introduce a new HEMA1 knockout mutant which grows only heterotrophically on MS (Murashige and Skoog) medium at low light, indicating that the remaining GluTR2 does not sufficiently compensate for the extensive needs of metabolic precursors for Chl. While hema1 accumulates low amounts of Chl, it contains more than half of the wild-type heme content. The functional diversity of the two GluTR isoforms was analyzed by means of complementation studies of the hema1 mutant by expression of pHEMA1::HEMA2 and p35S::HEMA1, respectively. Expression of both transgenes complements hema1, indicating that GluTR2 can likewise be involved in the synthesis of Chl and is not exclusively assigned to heme synthesis. In comparison with p35S::HEMA1-complemented hema1 and the wild type, GluTR2 expression under control of the HEMA1 promoter (pHEMA1) in pHEMA1::HEMA2-complemented hema1 mutants causes elevated protochlorophyllide levels under extended dark periods as well as in short-day-grown adult plants, resulting in the formation of necrotic leaf tissue. Although both GluTR isoforms have similar activity and contribute to 5-aminolevulinic acid synthesis for adequate accumulation of Chl and heme, it is proposed that the two proteins experience a different post-translational control in darkness and light. While GluTR2 continues 5-aminolevulinic acid synthesis in darkness, GluTR1 is efficiently inactivated by the interaction with the FLU (FLUORESCENT) protein, thereby preventing an accumulation of protochlorophyllide.

Biparental inheritance of organelles in Pelargonium: evidence for intergenomic recombination of mitochondrial DNA.

While uniparental transmission of mtDNA is widespread and dominating in eukaryotes leaving mutation as the major source of genotypic diversity, recently, biparental inheritance of mitochondrial genes has been demonstrated in reciprocal crosses of Pelargonium zonale and P. inquinans. The thereby arising heteroplasmy carries the potential for recombination between mtDNAs of different descent, i.e. between the parental mitochondrial genomes. We have analyzed these Pelargonium hybrids for mitochondrial intergenomic recombination events by examining differences in DNA blot hybridization patterns of the mitochondrial genes atp1 and cob. Further investigation of these genes and their flanking regions using nucleotide sequence polymorphisms and PCR revealed DNA segments in the progeny, which contained both P. zonale and P. inquinans sequences suggesting an intergenomic recombination in hybrids of Pelargonium. This turns Pelargonium into an interesting subject for studies of recombination and evolutionary dynamics of mitochondrial genomes.

Biparental inheritance of plastidial and mitochondrial DNA and hybrid variegation in Pelargonium.

Plastidial (pt) and mitochondrial (mt) genes usually show maternal inheritance. Non-Mendelian, biparental inheritance of plastids was first described by Baur (Z Indukt Abstamm Vererbungslehre 1:330-351, 1909) for crosses between Pelargonium cultivars. We have analyzed the inheritance of pt and mtDNA by examining the progeny from reciprocal crosses of Pelargonium zonale and P. inquinans using nucleotide sequence polymorphisms of selected pt and mt genes. Sequence analysis of the progeny revealed biparental inheritance of both pt and mtDNA. Hybrid plants exhibited variegation: our data demonstrate that the inquinans chloroplasts, but not the zonale chloroplasts bleach out, presumably due to incompatibility of the former with the hybrid nuclear genome. Different distribution of maternal and paternal sequences could be observed in different sectors of the same leaf, in different leaves of the same plant, and in different plants indicating random segregation and sorting-out of maternal and paternal plastids and mitochondria in the hybrids. The substantial transmission of both maternal and paternal mitochondria to the progeny turns Pelargonium into a particular interesting subject for studies on the inheritance, segregation and recombination of mt genes.