Jesse D. Woodson

Coordination of gene expression between organellar and nuclear genomes

Following the acquisition of chloroplasts and mitochondria by eukaryotic cells during endosymbiotic evolution, most of the genes in these organelles were either lost or transferred to the nucleus. Encoding organelle-destined proteins in the nucleus allows for host control of the organelle. In return, organelles send signals to the nucleus to coordinate nuclear and organellar activities. In photosynthetic eukaryotes, additional interactions exist between mitochondria and chloroplasts. Here we review recent advances in elucidating the intracellular signalling pathways that coordinate gene expression between organelles and the nucleus, with a focus on photosynthetic plants.

Heme Synthesis by Plastid Ferrochelatase I Regulates Nuclear Gene Expression in Plants

Chloroplast signals regulate hundreds of nuclear genes during development and in response to stress, but little is known of the signals or signal transduction mechanisms of plastid-to-nucleus (retrograde) signaling. In Arabidopsis thaliana, genetic studies using norflurazon (NF), an inhibitor of carotenoid biosynthesis, have identified five GUN (genomes uncoupled) genes, implicating the tetrapyrrole pathway as a source of a retrograde signal. Loss of function of any of these GUN genes leads to increased expression of photosynthesis-associated nuclear genes (PhANGs) when chloroplast development has been blocked by NF. Here we present a new Arabidopsis gain-of-function mutant, gun6-1D, with a similar phenotype. The gun6-1D mutant overexpresses the conserved plastid ferrochelatase 1 (FC1, heme synthase). Genetic and biochemical experiments demonstrate that increased flux through the heme branch of the plastid tetrapyrrole biosynthetic pathway increases PhANG expression. The second conserved plant ferrochelatase, FC2, colocalizes with FC1, but FC2 activity is unable to increase PhANG expression in undeveloped plastids. These data suggest a model in which heme, specifically produced by FC1, may be used as a retrograde signal to coordinate PhANG expression with chloroplast development.

Ubiquitin facilitates a quality-control pathway that removes damaged chloroplasts

Quality control one chloroplast at a time How do plant cells get rid of chloroplasts that are not working as they should? Woodson et al. describe a chloroplast quality-control pathway that allows for the selective elimination of individual chloroplasts. Damage by reactive oxygen species during photosynthesis is recognized by a ubiquitin ligase, which marks out damaged chloroplasts for degradation. The findings reveal how cells balance inherently stressful energy production with organelle turnover. Science, this issue p. 450 Singlet oxygen accumulation marks severely stressed chloroplasts for degradation. Energy production by chloroplasts and mitochondria causes constant oxidative damage. A functioning photosynthetic cell requires quality-control mechanisms to turn over and degrade chloroplasts damaged by reactive oxygen species (ROS). Here, we generated a conditionally lethal Arabidopsis mutant that accumulated excess protoporphyrin IX in the chloroplast and produced singlet oxygen. Damaged chloroplasts were subsequently ubiquitinated and selectively degraded. A genetic screen identified the plant U-box 4 (PUB4) E3 ubiquitin ligase as being necessary for this process. pub4-6 mutants had defects in stress adaptation and longevity. Thus, we have identified a signal that leads to the targeted removal of ROS-overproducing chloroplasts.

Organelle Signaling: How Stressed Chloroplasts Communicate with the Nucleus

Plastids are able to relay information to the nucleus to regulate stress responses. A new genetic screen has identified an isoprenoid intermediate that accumulates in stressed plastids and acts as a novel retrograde signal.

ABC Transporter for Corrinoids in Halobacterium sp. Strain NRC-1

We report evidence for the existence of a putative ABC transporter for corrinoid utilization in the extremely halophilic archaeon Halobacterium sp. strain NRC-1. Results from genetic and nutritional analyses of Halobacterium showed that mutants with lesions in open reading frames (ORFs) Vng1370G, Vng1371Gm, and Vng1369G required a 10(5)-fold higher concentration of cobalamin for growth than the wild-type or parent strain. The data support the conclusion that these ORFs encode orthologs of the bacterial cobalamin ABC transporter permease (btuC; Vng1370G), ATPase (btuD; Vng1371Gm), and substrate-binding protein (btuF; Vng1369G) components. Mutations in the Vng1370G, Vng1371Gm, and Vng1369G genes were epistatic, consistent with the hypothesis that their products work together to accomplish the same function. Extracts of btuF mutant strains grown in the presence of cobalamin did not contain any cobalamin molecules detectable by a sensitive bioassay, whereas btuCD mutant strain extracts did. The data are consistent with the hypothesis that the BtuF protein is exported to the extracellular side of the cell membrane, where it can bind cobalamin in the absence of BtuC and BtuD. Our data also provide evidence for the regulation of corrinoid transport and biosynthesis. Halobacterium synthesized cobalamin in a chemically defined medium lacking corrinoid precursors. To the best of our knowledge, this is the first genetic analysis of an archaeal corrinoid transport system.

The cbiS Gene of the Archaeon Methanopyrus kandleri AV19 Encodes a Bifunctional Enzyme with Adenosylcobinamide Amidohydrolase and α-Ribazole-Phosphate Phosphatase Activities

Here we report the initial biochemical characterization of the bifunctional alpha-ribazole-P (alpha-RP) phosphatase, adenosylcobinamide (AdoCbi) amidohydrolase CbiS enzyme from the hyperthermophilic methanogenic archaeon Methanopyrus kandleri AV19. The cbiS gene encodes a 39-kDa protein with two distinct segments, one of which is homologous to the AdoCbi amidohydrolase (CbiZ, EC 3.5.1.90) enzyme and the other of which is homologous to the recently discovered archaeal alpha-RP phosphatase (CobZ, EC 3.1.3.73) enzyme. CbiS function restored AdoCbi salvaging and alpha-RP phosphatase activity in strains of the bacterium Salmonella enterica where either step was blocked. The two halves of the cbiS genes retained their function in vivo when they were cloned separately. The CbiS enzyme was overproduced in Escherichia coli and was isolated to >95% homogeneity. High-performance liquid chromatography, UV-visible spectroscopy, and mass spectroscopy established alpha-ribazole and cobyric acid as the products of the phosphatase and amidohydrolase reactions, respectively. Reasons why the CbiZ and CobZ enzymes are fused in some archaea are discussed.

The Thiamine Kinase (YcfN) Enzyme Plays a Minor but Significant Role in Cobinamide Salvaging in Salmonella enterica

Cobinamide (Cbi) salvaging is impaired, but not abolished, in a Salmonella enterica strain lacking a functional cobU gene. CobU is a bifunctional enzyme (NTP:adenosylcobinamide [NTP:AdoCbi] kinase, GTP:adenosylcobinamide-phosphate [GTP:AdoCbi-P] guanylyltransferase) whose AdoCbi kinase activity is necessary for Cbi salvaging in this bacterium. Inactivation of the ycfN gene in a DeltacobU strain abrogated Cbi salvaging. Introduction of a plasmid carrying the ycfN(+) allele into a DeltacobU DeltaycfN strain substantially restored Cbi salvaging. Mass spectrometry data indicate that when YcfN-enriched cell extracts were incubated with AdoCbi and ATP, the product of the reaction was AdoCbi-P. Results from bioassays confirmed that YcfN converted AdoCbi to AdoCbi-P in an ATP-dependent manner. YcfN is a good example of enzymes that are used by the cell in multiple pathways to ensure the salvaging of valuable precursors.