Arabidopsis thaliana AtHAL3: a flavoprotein related to salt and osmotic tolerance and plant growth

The 26S proteasome is a multi-subunit ATP-dependent protease responsible for degrading most short-lived intracellular proteins targeted for breakdown by ubiquitin conjugation. The complex is composed of two relatively stable subparticles, the 20S proteasome, a hollow cylindrical structure which contains the proteolytic active sites in its lumen, and the 19S regulatory particle (RP) which binds to either end of the cylinder and provides the ATP-dependence and the specificity for ubiquitinated proteins. Among the approximately 18 subunits of the RP from yeast and animals are a set of six proteins, designated RPT1-6 for regulatory particle triple-A ATPase, that form a distinct family within the AAA superfamily. Presumably, these subunits use ATP hydrolysis to help assemble the 26S holocomplex, recognize and unfold appropriate substrates, and/or translocate the substrates to the 20S complex for degradation. Here, we describe the RPT gene family from Arabidopsis thaliana. From a collection of cDNAs and genomic sequences, a family of genes encoding all six of the RPT subunits was identified with significant amino acid sequence similarity to their yeast and animal counterparts. Five of the six RPT sub- units are encoded by two genes; the exception being RPT3 which is encoded by a single gene. mRNA for each of the six proteins is present in all tissue types examined. Five of the subunits (RPT1 and 3-6) complemented yeast mutants missing their respective orthologs, indicating that the yeast and Arabidopsis proteins are functionally equivalent. Taken together, these results demonstrate that the RP, like the 20S proteasome, is functionally and structurally conserved among eukaryotes and indicate that the plant RPT subunits, like their yeast counterparts, have non-redundant functions.     

The products of the mitochondrial orf25 and orfB genes are FO components in the plant F1FO ATP synthase

The F_O portion of the mitochondrial ATP synthase contains a range of different subunits in bacteria, yeast and mammals. A search of the Arabidopsis genome identified sequence orthologs for only some of these subunits. Blue native polyacrylamide gel electrophoresis separation of Arabidopsis mitochondrial respiratory chain complexes revealed intact F₁F_O, and separated F₁ and F_O components. The subunits of each complex were analysed by mass spectrometry and matched to Arabidopsis genes. In the F₁F_O complex a series of nine known subunits were identified along with two additional proteins matching the predicted products of the mitochondrial encoded orfB and orf25 genes. The F₁ complex contained the five well-characterised F₁ subunits, while four subunits in the F_O complex were identified: subunit 9, d subunit, and the orfB and orf25 products. Previously, orfB has been suggested as the plant equivalent of subunit 8 based on structural and sequence similarity. We propose that orf25 is the plant b subunit based on structural similarity and its presence in the F_O complex. Chimerics of orf25, orfB, subunit 9 and subunit 6 have been associated with cytoplasmic male sterility in a variety of plant species, our additional findings now place all these proteins in the same protein complex.     

Arabidopsis vacuolar H+-ATPase (V-ATPase) B subunits are involved in actin cytoskeleton remodeling via binding to, bundling, and stabilizing F-actin

Vacuolar H(+)-ATPase (V-ATPase) is a membrane-bound multisubunit enzyme complex composed of at least 14 different subunits. The complex regulates the physiological processes of a cell by controlling the acidic environment, which is necessary for certain activities and the interaction with the actin cytoskeleton through its B and C subunits in both humans and yeast. Arabidopsis V-ATPase has three B subunits (AtVAB1, AtVAB2, and AtVAB3), which share 97.27% sequence identity and have two potential actin-binding sites, indicating that these AtVABs may have crucial functions in actin cytoskeleton remodeling and plant cell development. However, their biochemical functions are poorly understood. In this study, we demonstrated that AtVABs bind to and co-localize with F-actin, bundle F-actin to form higher order structures, and stabilize actin filaments in vitro. In addition, the AtVABs also show different degrees of activities in capping the barbed ends but no nucleating activities, and these activities were not regulated by calcium. The functional similarity and differences of the AtVABs implied that they may play cooperative and distinct roles in Arabidopsis cells.     

Human orthologs of yeast vacuolar protein sorting proteins Vps26, 29, and 35: assembly into multimeric complexes

Sorting nexin (SNX) 1 and SNX2 are mammalian orthologs of Vps5p, a yeast protein that is a subunit of a large multimeric complex, termed the retromer complex, involved in retrograde transport of proteins from endosomes to the trans-Golgi network. We report the cloning and characterization of human orthologs of three additional components of the complex: Vps26p, Vps29p, and Vps35p. The close structural similarity between the yeast and human proteins suggests a similarity in function. We used both yeast two-hybrid assays and expression in mammalian cells to define the binding interactions among these proteins. The data suggest a model in which hVps35 serves as the core of a multimeric complex by binding directly to hVps26, hVps29, and SNX1. Deletional analyses of hVps35 demonstrate that amino acid residues 1-53 and 307-796 of hVps35 bind to the coiled coil-containing domain of SNX1. In contrast, hVps26 binds to amino acid residues 1-172 of hVps35, whereas hVps29 binds to amino acid residues 307-796 of hVps35. Furthermore, hVps35, hVps29, and hVps26 have been found in membrane-associated and cytosolic compartments. Gel filtration chromatography of COS7 cell cytosol showed that both recombinant and endogenous hVps35, hVps29, and hVps26 coelute as a large complex ( approximately 220-440 kDa). In the absence of hVps35, neither hVps26 nor hVps29 is found in the large complex. These data provide the first insights into the binding interactions among subunits of a putative mammalian retromer complex.     

Functional divergence of a syntenic invertase gene family in tomato,potato, and Arabidopsis

Comparative analysis of complex developmental pathways depends on our ability to resolve the function of members of gene families across taxonomic groups. LIN5, which belongs to a small gene family of apoplastic invertases in tomato (Lycopersicon esculentum), is a quantitative trait locus that modifies fruit sugar composition. We have compared the genomic organization and expression of this gene family in the two distantly related species: tomato and Arabidopsis. Invertase family members reside on segmental duplications in the near-colinear genomes of tomato and potato (Solanum tuberosum). These chromosomal segments are syntenically duplicated in the model plant Arabidopsis. On the basis of phylogenetic analysis of genes in the microsyntenic region, we conclude that these segmental duplications arose independently after the separation of the tomato/potato clade from Arabidopsis. Rapid regulatory divergence is characteristic of the invertase family. Interestingly, although the processes of gene duplication and specialization of expression occurred separately in the two species, synteny-based orthologs from both clades acquired similar organ-specific expression. This similar expression pattern of the genes is evidence of comparable evolutionary constraints (parallel evolution) rather than of functional orthology. The observation that functional orthology cannot be identified through analysis of expression similarity highlights the caution that needs to be exercised in extrapolating developmental networks from a model organism.     

Interactions between Transport Protein Particle (TRAPP) complexes and Rab GTPases in Arabidopsis

Transport Protein Particle II (TRAPPII) is essential for exocytosis, endocytosis, protein sorting and cytokinesis. In spite of a considerable understanding of its biological role, little information is known about Arabidopsis TRAPPII complex topology and molecular function. In this study, independent proteomic approaches initiated with TRAPP components or Rab‐A GTPase variants converge on the TRAPPII complex. We show that the Arabidopsis genome encodes the full complement of 13 TRAPPC subunits, including four previously unidentified components. A dimerization model is proposed to account for binary interactions between TRAPPII subunits. Preferential binding to dominant negative (GDP‐bound) versus wild‐type or constitutively active (GTP‐bound) RAB‐A2a variants discriminates between TRAPPII and TRAPPIII subunits and shows that Arabidopsis complexes differ from yeast but resemble metazoan TRAPP complexes. Analyzes of Rab‐A mutant variants in trappii backgrounds provide genetic evidence that TRAPPII functions upstream of RAB‐A2a, allowing us to propose that TRAPPII is likely to behave as a guanine nucleotide exchange factor (GEF) for the RAB‐A2a GTPase. GEFs catalyze exchange of GDP for GTP; the GTP‐bound, activated, Rab then recruits a diverse local network of Rab effectors to specify membrane identity in subsequent vesicle fusion events. Understanding GEF?Rab interactions will be crucial to unravel the co‐ordination of plant membrane traffic.     

Molecular and functional analysis of phosphomannomutase (PMM) from higher plants and genetic evidence for the involvement of PMM in ascorbic acid biosynthesis in Arabidopsis and Nicotiana benthamiana

Phosphomannomutase (PMM) catalyzes the interconversion of mannose-6-phosphate and mannose-1-phosphate. However, systematic molecular and functional investigations on PMM from higher plants have hitherto not been reported. In this work, PMM cDNAs were isolated from Arabidopsis, Nicotiana benthamiana, soybean, tomato, rice and wheat. Amino acid sequence comparisons indicated that plant PMM proteins exhibited significant identity to their fungal and mammalian orthologs. In line with the similarity in primary structure, plant PMM complemented the sec53-6 temperature sensitive mutant of Saccharomyces cerevisiae. Histidine-tagged Arabidopsis PMM (AtPMM) purified from Escherichia coli converted mannose-1-phosphate into mannose-6-phosphate and glucose-1-phosphate into glucose-6-phosphate, with the former reaction being more efficient than the latter one. In Arabidopsis and N. benthamiana, PMM was constitutively expressed in both vegetative and reproductive organs. Reducing the PMM expression level through virus-induced gene silencing caused a substantial decrease in ascorbic acid (AsA) content in N. benthamiana leaves. Conversely, raising the PMM expression level in N. benthamiana using viral-vector-mediated ectopic expression led to a 20-50% increase in AsA content. Consistent with this finding, transgenic expression of an AtPMM-GFP fusion protein in Arabidopsis also increased AsA content by 25-33%. Collectively, this study improves our understanding on the molecular and functional properties of plant PMM and provides genetic evidence on the involvement of PMM in the biosynthesis of AsA in Arabidopsis and N. benthamiana plants.     

Characterization of the plant homologue of prohibitin,a gene associated with antiproliferative activity in mammalian cells

This report describes the cloning and characterization of a plant cDNA coding for a protein which shows high amino acid sequence similarity with prohibitin, whose gene is associated with antiproliferative activity in mammalian cells. Arabidopsis thaliana and Nicotiana tabacum prohibitin complete cDNAs were isolated, and the expression pattern of prohibitin was examined using polyclonal antibodies raised against the Arabidopsis recombinant prohibitin expressed in Escherichia coli. A single immunoreactive protein was detected in various plant species and in all Arabidopsis organs examined. Subcellular fractionation using tobacco leaves revealed prohibitin in a mitochondrial-enriched fraction. Phylogenetic conservation of prohibitin's amino acid sequence and subcellular localization suggests a similar function in plants, yeast and mammals.     

Structure and functional analyses of the 26S proteasome subunits from plants – Plant 26S proteasome

As initial steps to define how the 26S proteasome degrades ubiquitinated proteins in plants, we have characterized many of the subunits that comprise the proteolytic complex from Arabidopsis thaliana. A set of 23 Arabidopsis genes encoding the full complement of core particle (CP) subunits and a collection encoding 12 out of 18 known eukaryotic regulatory particle (RP) subunits, including six AAA-ATPase subunits, were identified. Several of these 26S proteasome genes could complement yeast strains missing the corresponding orthologs. Using this ability of plant subunits to functionally replace yeast counterparts, a parallel structure/function analysis was performed with the RP subunit RPN10/MCB1, a putative receptor for ubiquitin conjugates. RPN10 is not essential for yeast viability but is required for amino acid analog tolerance and degradation of proteins via the ubiquitin-fusion degradation pathway, a subpathway within the ubiquitin system. Surprisingly, we found that the C-terminal motif required for conjugate recognition by RPN10 is not essential for in vivo functions. Instead, a domain near the N-terminus is required. We have begun to exploit the moss Physcomitrella patens as a model to characterize the plant 26S proteasome using reverse genetics. By homologous recombination, we have successfully disrupted the RPN10 gene. Unlike yeast rpn10 strains which grow normally, Physcomitrella rpn10 strains are developmentally arrested, being unable to initiate gametophorogenesis. Further analysis of these mutants revealed that RPN10 is likely required for a developmental program triggered by plant hormones.