Respiratory complex I deficiency induces drought tolerance by impacting leaf stomatal and hydraulic conductances

To investigate the role of plant mitochondria in drought tolerance, the response to water deprivation was compared between Nicotiana sylvestris wild type (WT) plants and the CMSII respiratory complex I mutant, which has low-efficient respiration and photosynthesis, high levels of amino acids and pyridine nucleotides, and increased antioxidant capacity. We show that the delayed decrease in relative water content after water withholding in CMSII, as compared to WT leaves, is due to a lower stomatal conductance. The stomatal index and the abscisic acid (ABA) content were unaffected in well-watered mutant leaves, but the ABA/stomatal conductance relation was altered during drought, indicating that specific factors interact with ABA signalling. Leaf hydraulic conductance was lower in mutant leaves when compared to WT leaves and the role of oxidative aquaporin gating in attaining a maximum stomatal conductance is discussed. In addition, differences in leaf metabolic status between the mutant and the WT might contribute to the low stomatal conductance, as reported for TCA cycle-deficient plants. After withholding watering, TCA cycle derived organic acids declined more in CMSII leaves than in the WT, and ATP content decreased only in the CMSII. Moreover, in contrast to the WT, total free amino acid levels declined whilst soluble protein content increased in CMSII leaves, suggesting an accelerated amino acid remobilisation. We propose that oxidative and metabolic disturbances resulting from remodelled respiration in the absence of Complex I activity could be involved in bringing about the lower stomatal and hydraulic conductances.     

Three new BLM gene mutations associated with Bloom syndrome

Bloom's syndrome (BS) is a rare autosomal recessive disease predisposing patients to all types of cancers affecting the general population. BS cells display a high level of genetic instability, including a 10-fold increase in the rate of sister chromatid exchanges, currently the only objective criterion for BS diagnosis. We have developed a method for screening the BLM gene for mutations based on direct genomic DNA sequencing. A questionnaire based on clinical information, cytogenetic features, and family history was addressed to physicians prescribing BS genetic screening, with the aim of confirming or guiding diagnosis. We report here four BLM gene mutations, three of which have not been described before. Three of the mutations are frameshift mutations, and the fourth is a nonsense mutation. All these mutations introduce a stop codon, and may therefore be considered to have deleterious biological effect. This approach should make it possible to identify new mutations and to correlate them with clinical information.     

NPI1, an essential yeast gene involved in induced degradation of Gap1 and Fur4 permeases, encodes the Rsp5 ubiquitin—protein ligase

When yeast cells growing on a poor nitrogen source are supplied with NH₄⁺ ions, several nitrogen permeases including the general amino acid permease (Gap1p) are rapidly and completely inactivated. This report shows that inactivation by NH₄⁺ of the Gap1 permease is accompanied by its degradation. A functional NPI1 gene product is required for both inactivation and degradation of Gap1p. Molecular analysis of the NPI1 gene showed that it is identical to RSP5 . The RSP5 product is a ubiquitin—protein ligase (E3 enzyme) whose physiological function was, however, unknown. Its C-terminal region is very similar to that of other members of the E6-AP-like family of ubiquitin-protein ligases. Its N-terminal region contains a single C₂ domain that may be a Ca²⁺-dependent phospholipid interaction motif, followed by several copies of a recently identified domain called WW(P). The Npi1/Rsp5 protein has a homologue both in humans and in mice, the latter being involved in brain development. Stress-induced degradation of the uracil permease (Fur4p), a process in which ubiquitin is probably involved, was also found to require a functional NPI1/RSP5 product. Chromosomal deletion of NPI1/RSP5 showed that this gene is essential for cell viability. In the viable np1/rsp5 strain, expression of NPI1/RSP5 is reduced as a result of insertion of a Ty1 element in its 5' region. Our results show that the Npi1/Rsp5 ubiquitin-protein ligase participates in induced degradation of at least two permeases, Gap1p and Fur4p, and probably also other proteins.     

Analysis of heme biosynthesis in catalase and cytochrome deficient yeast mutants

Summary Mutants of Saccharomyces cerevisiae, described as catalase and cytochromes deficient (Pachecka et al., 1974), have been analyzed for heme biosynthesis ability. Some enzymatic activities involved in protoheme synthesis were measured in acellular extracts, whereas whole cells were analyzed for cytochrome spectra and for possible accumulation of porphyrin synthesis intermediates. A good correlation was found between these in vitro and in vivo studies. Results show that two mutants were impaired in 5-aminolevulinate synthesis, two mutants were devoid of uroporphyrinogen I synthetase activity and one mutant presented defects in coproporphyrinogen III oxidase activity.     

Heterologous expression of a plant uracil transporter in yeast: improvement of plasma membrane targeting in mutants of the Rsp5p ubiquitin protein ligase

Plasma membrane proteins involved in transport processes play a crucial role in cell physiology. On account of these properties, these molecules are ideal targets for development of new therapeutic and agronomic agents. However, these proteins are of low abundance, which limits their study. Although yeast seems ideal for expressing heterologous transporters, plasma membrane proteins are often retained in intracellular compartments. We tried to find yeast mutants potentially able to improve functional expression of a whole set of heterologous transporters. We focused on Arabidopsis thaliana ureide transporter 1 (AtUPS1), previously cloned by functional complementation in yeast. Tagged versions of AtUPS1 remain mostly trapped in the endoplasmic reticulum and were able to reach slowly the plasma membrane. In contrast, untagged AtUPS1 is rapidly delivered to plasma membrane, where it remains in stable form. Tagged and untagged versions of AtUPS1 were expressed in cells deficient in the ubiquitin ligase Rsp5p, involved in various stages of the intracellular trafficking of membrane-bound proteins. rsp5 mutants displayed improved steady state amounts of untagged and tagged versions of AtUPS1. rsp5 cells are thus powerful tools to solve the many problems inherent to heterologous expression of membrane proteins in yeast, including ER retention.     

Yeast Rsp5 ubiquitin ligase affects the actin cytoskeleton in vivo and in vitro

Yeast Rsp5 ubiquitin ligase is involved in several cellular processes, including endocytosis. Actin patches are sites of endocytosis, a process involving actin assembly and disassembly. Here we show Rsp5 localization in cortical patches and demonstrate its involvement in actin cytoskeleton organization and dynamics. We found that the Rsp5-F1-GFP2 N-terminal fragment and full length GFP-Rsp5 were recruited to peripheral patches that temporarily co-localized with Abp1-mCherry, a marker of actin patches. Actin cytoskeleton organization was defective in a strain lacking RSP5 or overexpressing RSP5, and this phenotype was accompanied by morphological abnormalities. Overexpression of RSP5 caused hypersensitivity of cells to Latrunculin A, an actin-depolymerizing drug and was toxic to cells lacking Las17, an activator of actin nucleation. Moreover, Rsp5 was required for efficient actin polymerization in a whole cell extract based in vitro system. Rsp5 interacted with Las17 and Las17-binding proteins, Lsb1 and Lsb2, in a GST-Rsp5-WW2/3 pull down assay. Rsp5 ubiquitinated Lsb1-HA and Lsb2-HA without directing them for degradation. Overexpression of RSP5 increased the cellular level of HA-Las17 in wild type and in lsb1Δ lsb2Δ strains in which the basal level of Las17 was already elevated. This increase was prevented in a strain devoid of Las17-binding protein Sla1 which is also a target of Rsp5 ubiquitination. Thus, Rsp5 together with Lsb1, Lsb2 and Sla1 regulate the level of Las17, an important activator of actin polymerization.     

A perturbed ubiquitin landscape distinguishes between ubiquitin in trafficking and in proteolysis

Any of seven lysine residues on ubiquitin can serve as the base for chain-extension, resulting in a sizeable spectrum of ubiquitin modifications differing in chain length or linkage type. By optimizing a procedure for rapid lysis, we charted the profile of conjugated cellular ubiquitin directly from whole cell extract. Roughly half of conjugated ubiquitin (even at high molecular weights) was nonextended, consisting of monoubiquitin modifications and chain terminators (endcaps). Of extended ubiquitin, the primary linkages were via Lys48 and Lys63. All other linkages were detected, contributing a relatively small portion that increased at lower molecular weights. In vivo expression of lysineless ubiquitin (K0 Ub) perturbed the ubiquitin landscape leading to elevated levels of conjugated ubiquitin, with a higher mono-to-poly ratio. Affinity purification of these trapped conjugates identified a comprehensive list of close to 900 proteins including novel targets. Many of the proteins enriched by K0 ubiquitination were membrane-associated, or involved in cellular trafficking. Prime among them are components of the ESCRT machinery and adaptors of the Rsp5 E3 ubiquitin ligase. Ubiquitin chains associated with these substrates were enriched for Lys63 linkages over Lys48, indicating that K0 Ub is unevenly distributed throughout the ubiquitinome. Biological assays validated the interference of K0 Ub with protein trafficking and MVB sorting, minimally affecting Lys48-dependent turnover of proteasome substrates. We conclude that despite the shared use of the ubiquitin molecule, the two branches of the ubiquitin machinery--the ubiquitin-proteasome system and the ubiquitin trafficking system--were unevenly perturbed by expression of K0 ubiquitin.     

L-galactono-1,4-lactone dehydrogenase is required for the accumulation of plant respiratory complex I

Mitochondrial NADH-ubiquinone oxidoreductase (complex I) is the largest enzyme of the oxidative phosphorylation system, with subunits located at the matrix and membrane domains. In plants, holocomplex I is composed of more than 40 subunits, 9 of which are encoded by the mitochondrial genome (NAD subunits). In Nicotiana sylvestris, a minor 800-kDa subcomplex containing subunits of both domains and displaying NADH dehydrogenase activity is detectable. The NMS1 mutant lacking the membrane arm NAD4 subunit and the CMSII mutant lacking the peripheral NAD7 subunit are both devoid of the holoenzyme. In contrast to CMSII, the 800-kDa subcomplex is present in NMS1 mitochondria, indicating that it could represent an assembly intermediate lacking the distal part of the membrane arm. L-galactono-1,4-lactone dehydrogenase (GLDH), the last enzyme in the plant ascorbate biosynthesis pathway, is associated with the 800-kDa subcomplex but not with the holocomplex. To investigate possible relationships between GLDH and complex I assembly, we characterized an Arabidopsis thaliana gldh insertion mutant. Homozygous gldh mutant plants were not viable in the absence of ascorbate supplementation. Analysis of crude membrane extracts by blue native and two-dimensional SDS-PAGE showed that complex I accumulation was strongly prevented in leaves and roots of Atgldh plants, whereas other respiratory complexes were found in normal amounts. Our results demonstrate the role of plant GLDH in both ascorbate biosynthesis and complex I accumulation.