Arabidopsis membrane-anchored ubiquitin-fold (MUB) proteins localize a specific subset of ubiquitin-conjugating (E2) enzymes to the plasma membrane.

The covalent attachment of ubiquitin (Ub) to various intracellular proteins plays important roles in altering the function, localization, processing, and degradation of the modified target. A minimal ubiquitylation pathway uses a three-enzyme cascade (E1, E2, and E3) to activate Ub and select target proteins for modification. Although diverse E3 families provide much of the target specificity, several factors have emerged recently that coordinate the subcellular localization of the ubiquitylation machinery. Here, we show that the family of membrane-anchored ubiquitin-fold (MUB) proteins recruits and docks specific E2s to the plasma membrane. Protein interaction screens with Arabidopsis MUBs revealed that interacting E2s are limited to a well defined subgroup that is phylogenetically related to human UbcH5 and yeast Ubc4/5 families. MUBs appear to interact noncovalently with an E2 surface opposite the active site that forms a covalent linkage with Ub. Bimolecular fluorescence complementation demonstrated that MUBs bind simultaneously to the plasma membrane via a prenyl tail and to the E2 in planta. These findings suggest that MUBs contribute subcellular specificity to ubiquitylation by docking the conjugation machinery to the plasma membrane.

Tandem affinity purification and mass spectrometric analysis of ubiquitylated proteins in Arabidopsis.

Protein ubiquitylation is a central regulatory mechanism that controls numerous processes in plants, including hormone signaling, developmental progression, responses to biotic and abiotic challenges, protein trafficking and chromatin structure. Despite data implicating thousands of plant proteins as targets, so far only a few have been conclusively shown to be ubiquitylated in planta. Here we describe a method to isolate ubiquitin-protein conjugates from Arabidopsis that exploits a stable transgenic line expressing a synthetic poly-UBQ gene encoding ubiquitin (Ub) monomers N-terminally tagged with hexahistidine. Following sequential enrichment by Ub-affinity and nickel chelate-affinity chromatography, the ubiquitylated proteins were trypsinized, separated by two-dimensional liquid chromatography, and analyzed by mass spectrometry. Our list of 54 non-redundant targets, expressed by as many as 90 possible isoforms, included those predicted by genetic studies to be ubiquitylated in plants (EIN3 and JAZ6) or shown to be ubiquitylated in other eukaryotes (ribosomal subunits, elongation factor 1alpha, histone H1, HSP70 and CDC48), as well as candidates whose control by the Ub/26S proteasome system is not yet appreciated. Ub attachment site(s) were resolved for a subset of these proteins, but surprisingly little sequence consensus was detected, implying that specific residues surrounding the modified lysine are not important determinants for ubiquitylation. We also identified six of the seven available lysine residues on Ub itself as Ub attachment sites, together with evidence for a branched mixed-linkage chain, suggesting that the topologies of Ub chains can be highly complex in plants. Taken together, our method provides a widely applicable strategy to define ubiquitylation in any tissue of intact plants exposed to a wide range of conditions.

Translocation and assembly of mitochondrially coded Saccharomyces cerevisiae cytochrome c oxidase subunit Cox2 by Oxa1 and Yme1 in the absence of Cox18.

Members of the Oxa1/YidC/Alb3 family of protein translocases are essential for assembly of energy-transducing membrane complexes. In Saccharomyces cerevisiae, Oxa1 and its paralog, Cox18, are required for assembly of Cox2, a mitochondrially encoded subunit of cytochrome c oxidase. Oxa1 is known to be required for cotranslational export of the Cox2 N-terminal domain across the inner mitochondrial membrane, while Cox18 is known to be required for post-translational export of the Cox2 C-tail domain. We find that overexpression of Oxa1 does not compensate for the absence of Cox18 at the level of respiratory growth. However, it does promote some translocation of the Cox2 C-tail domain across the inner membrane and causes increased accumulation of Cox2, which remains unassembled. This result suggests that Cox18 not only translocates the C-tail, but also must deliver it in a distinct state competent for cytochrome oxidase assembly. We identified respiring mutants from a cox18Delta strain overexpressing OXA1, whose respiratory growth requires overexpression of OXA1. The recessive nuclear mutations allow some assembly of Cox2 into cytochrome c oxidase. After failing to identify these mutations by methods based on transformation, we successfully located them to MGR1 and MGR3 by comparative hybridization to whole-genome tiling arrays and microarray-assisted bulk segregant analysis followed by linkage mapping. While Mgr1 and Mgr3 are known to associate with the Yme1 mitochondrial inner membrane i-AAA protease and to participate in membrane protein degradation, their absence does not appear to stabilize Cox2 under these conditions. Instead, Yme1 probably chaperones the folding and/or assembly of Oxa1-exported Cox2 in the absence of Mrg1 or Mgr3, since respiratory growth and cytochrome c oxidase assembly in a cox18 mgr3 double-mutant strain overexpressing OXA1 is YME1 dependent.

Introduction of cytochrome b mutations in Saccharomyces cerevisiae by a method that allows selection for both functional and non-functional cytochrome b proteins.

We have previously used inhibitors interacting with the Qn site of the yeast cytochrome bc(1) complex to obtain yeast strains with resistance-conferring mutations in cytochrome b as a means to investigate the effects of amino acid substitutions on Qn site enzymatic activity [M.G. Ding, J.-P. di Rago, B.L. Trumpower, Investigating the Qn site of the cytochrome bc1 complex in Saccharomyces cerevisiae with mutants resistant to ilicicolin H, a novel Qn site inhibitor, J. Biol. Chem. 281 (2006) 36036-36043.]. Although the screening produced various interesting cytochrome b mutations, it depends on the availability of inhibitors and can only reveal a very limited number of mutations. Furthermore, mutations leading to a respiratory deficient phenotype remain undetected. We therefore devised an approach where any type of mutation can be efficiently introduced in the cytochrome b gene. In this method ARG8, a gene that is normally encoded by nuclear DNA, replaces the naturally occurring mitochondrial cytochrome b gene, resulting in ARG8 expressed from the mitochondrial genome (ARG8(m)). Subsequently replacing ARG8(m) with mutated versions of cytochrome b results in arginine auxotrophy. Respiratory competent cytochrome b mutants can be selected directly by virtue of their ability to restore growth on non-fermentable substrates. If the mutated cytochrome b is non-functional, the presence of the COX2 respiratory gene marker on the mitochondrial transforming plasmid enables screening for cytochrome b mutants with a stringent respiratory deficiency (mit(-)). With this system, we created eight different yeast strains containing point mutations at three different codons in cytochrome b affecting center N. In addition, we created three point mutations affecting arginine 79 in center P. This is the first time mutations have been created for three of the loci presented here, and nine of the resulting mutants have never been described before.

Cox18p is required for export of the mitochondrially encoded Saccharomyces cerevisiae Cox2p C-tail and interacts with Pnt1p and Mss2p in the inner membrane.

The amino- and carboxy-terminal domains of mitochondrially encoded cytochrome c oxidase subunit II (Cox2p) are translocated out of the matrix to the intermembrane space. We have carried out a genetic screen to identify components required to export the biosynthetic enzyme Arg8p, tethered to the Cox2p C terminus by a translational gene fusion inserted into mtDNA. We obtained multiple alleles of COX18, PNT1, and MSS2, as well as mutations in CBP1 and PET309. Focusing on Cox18p, we found that its activity is required to export the C-tail of Cox2p bearing a short C-terminal epitope tag. This is not a consequence of reduced membrane potential due to loss of cytochrome oxidase activity because Cox2p C-tail export was not blocked in mitochondria lacking Cox4p. Cox18p is not required to export the Cox2p N-tail, indicating that these two domains of Cox2p are translocated by genetically distinct mechanisms. Cox18p is a mitochondrial integral inner membrane protein. The inner membrane proteins Mss2p and Pnt1p both coimmunoprecipitate with Cox18p, suggesting that they work together in translocation of Cox2p domains, an inference supported by functional interactions among the three genes.

Interactions among COX1, COX2, and COX3 mRNA-specific translational activator proteins on the inner surface of the mitochondrial inner membrane of Saccharomyces cerevisiae.

The core of the cytochrome c oxidase complex is composed of its three largest subunits, Cox1p, Cox2p, and Cox3p, which are encoded in mitochondrial DNA of Saccharomyces cerevisiae and inserted into the inner membrane from the inside. Mitochondrial translation of the COX1, COX2, and COX3 mRNAs is activated mRNA specifically by the nuclearly coded proteins Pet309p, Pet111p, and the concerted action of Pet54p, Pet122p, and Pet494p, respectively. Because the translational activators recognize sites in the 5'-untranslated leaders of these mRNAs and because untranslated mRNA sequences contain information for targeting their protein products, the activators are likely to play a role in localizing translation. Herein, we report physical associations among the mRNA-specific translational activator proteins, located on the matrix side of the inner membrane. These interactions, detected by coimmune precipitation and by two-hybrid experiments, suggest that the translational activator proteins could be organized on the surface of the inner membrane such that synthesis of Cox1p, Cox2p, and Cox3p would be colocalized in a way that facilitates assembly of the core of the cytochrome c oxidase complex. In addition, we found interactions between Nam1p/Mtf2p and the translational activators, suggesting an organized delivery of mitochondrial mRNAs to the translation system.

Multiple roles of the Cox20 chaperone in assembly of Saccharomyces cerevisiae cytochrome c oxidase.

The Cox2 subunit of Saccharomyces cerevisiae cytochrome c oxidase is synthesized in the mitochondrial matrix as a precursor whose leader peptide is rapidly processed by the inner membrane protease following translocation to the intermembrane space. Processing is chaperoned by Cox20, an integral inner membrane protein whose hydrophilic domains are located in the intermembrane space, and Cox20 remains associated with mature, unassembled Cox2. The Cox2 C-tail domain is exported post-translationally by the highly conserved translocase Cox18 and associated proteins. We have found that Cox20 is required for efficient export of the Cox2 C-tail. Furthermore, Cox20 interacts by co-immune precipitation with Cox18, and this interaction requires the presence of Cox2. We therefore propose that Cox20 binding to Cox2 on the trans side of the inner membrane accelerates dissociation of newly exported Cox2 from the Cox18 translocase, promoting efficient cycling of the translocase. The requirement for Cox20 in cytochrome c oxidase assembly and respiratory growth is partially bypassed by yme1, mgr1 or mgr3 mutations, each of which reduce i-AAA protease activity in the intermembrane space. Thus, Cox20 also appears to stabilize unassembled Cox2 against degradation by the i-AAA protease. Pre-Cox2 leader peptide processing by Imp1 occurs in the absence of Cox20 and i-AAA protease activity, but is greatly reduced in efficiency. Under these conditions some mature Cox2 is assembled into cytochrome c oxidase allowing weak respiratory growth. Thus, the Cox20 chaperone has important roles in leader peptide processing, C-tail export, and stabilization of Cox2.

Chapter 27 An improved method for introducing point mutations into the mitochondrial cytochrome B gene to facilitate studying the role of cytochrome B in the formation of reactive oxygen species.

Cytochrome b is a pivotal protein subunit of the cytochrome bc(1) complex and forms the ubiquinol oxidation site in the enzyme that is generally thought to be the primary site where electrons are aberrantly diverted from the enzyme, reacting with oxygen to form superoxide anion. In addition, recent studies have shown that mutations in cytochrome b can substantially increase rates of oxygen radical formation by the bc(1) complex. It would, thus, be advantageous to be able to manipulate cytochrome b by mutagenesis of the cytochrome b gene to better understand the role of cytochrome b in oxygen radical formation. Cytochrome b is encoded in the mitochondrial genome in eukaryotic cells, and introduction of point mutations into the gene is generally cumbersome because of the tedious screening process for positive clones. In addition, previously it has been especially difficult to introduce point mutations that lead to loss of respiratory function, as might be expected of mutations that markedly enhance oxygen radical formation. To more efficiently introduce amino acid changes into cytochrome b we have devised a method for mutagenesis of the Saccharomyces cerevisiae mitochondrial cytochrome b gene that uses a recoded ARG8 gene as a "placeholder" for the wild-type b gene. In this method ARG8, a gene that is normally encoded by nuclear DNA, replaces the naturally occurring mitochondrial cytochrome b gene, resulting in ARG8 expressed from the mitochondrial genome (ARG8(m)). Subsequently replacing ARG8(m) with mutated versions of cytochrome b results in arginine auxotrophy. Respiratory-competent cytochrome b mutants can be selected directly by virtue of their ability to restore growth on nonfermentable substrates. If the mutated cytochrome b is nonfunctional, the presence of the COX2 respiratory gene marker on the mitochondrial transforming plasmid enables screening for cytochrome b mutants with a stringent respiratory deficiency (mit(-)).

Genetic analysis of SUMOylation in Arabidopsis: conjugation of SUMO1 and SUMO2 to nuclear proteins is essential.

The posttranslational addition of small ubiquitin-like modifiers (SUMOs) to other intracellular proteins has been implicated in a variety of eukaryotic functions, including modifying cytoplasmic signal transduction, nuclear import and subnuclear compartmentalization, DNA repair, and transcription regulation. For plants, in particular, both genetic analyses and the rapid accumulation of SUMO conjugates in response to various adverse environmental conditions suggest that SUMOylation plays a key role in the stress response. Through genetic analyses of various SUMO conjugation mutants, we show here that the SUMO1 and SUMO2 isoforms, in particular, and SUMOylation, in general, are essential for viability in Arabidopsis (Arabidopsis thaliana). Null T-DNA insertion mutants affecting the single genes encoding the SUMO-activating enzyme subunit SAE2 and the SUMO-conjugating enzyme SCE1 are embryonic lethal, with arrest occurring early in embryo development. Whereas the single genes encoding the SUMO1 and SUMO2 isoforms are not essential by themselves, double mutants missing both are also embryonic lethal. Viability can be restored by reintroduction of SUMO1 expression in the homozygous sum1-1 sum2-1 background. Various stresses, like heat shock, dramatically increase the pool of SUMO conjugates in planta. This increase involves SUMO1 and SUMO2 and is mainly driven by the SUMO protein ligase SIZ1, with most of the conjugates accumulating in the nucleus. Taken together, it appears that SIZ1-mediated conjugation of SUMO1 and SUMO2 to other intracellular proteins is essential in Arabidopsis, possibly through stress-induced modification of a potentially diverse pool of nuclear proteins.

MUBs, a family of ubiquitin-fold proteins that are plasma membrane-anchored by prenylation.

Ubiquitin (Ub)-fold proteins are rapidly emerging as an important class of eukaryotic modifiers, which often exert their influence by post-translational addition to other intracellular proteins. Despite assuming a common beta-grasp three-dimensional structure, their functions are highly diverse because of distinct surface features and targets and include tagging proteins for selective breakdown, nuclear import, autophagic recycling, vesicular trafficking, polarized morphogenesis, and the stress response. Here we describe a novel family of Membrane-anchored Ub-fold (MUB) proteins that are present in animals, filamentous fungi, and plants. Extending from the C terminus of the Ub-fold is typically a cysteine-containing CAAX (where A indicates aliphatic amino acid) sequence that can direct the attachment of either a 15-carbon farnesyl or a 20-carbon geranylgeranyl moiety in vitro. Modified forms of several MUBs were detected in transgenic Arabidopsis thaliana, suggesting that these MUBs are prenylated in vivo. Both cell fractionation and confocal microscopic analyses of Arabidopsis plants expressing GFP-MUB fusions showed that the modified forms are membrane-anchored with a significant enrichment on the plasma membrane. This plasma membrane location was blocked in vivo in prenyltransferase mutants and by mevinolin, which inhibits the synthesis of prenyl groups. In addition to the five MUBs with CAAX boxes, Arabidopsis has one MUB variant with a cysteine-rich C terminus distinct from the CAAX box that is also membrane-anchored, possibly through the attachment of a long chain acyl group. Although the physiological role(s) of MUBs remain unknown, the discovery of these prenylated forms further expands the diversity and potential functions of Ub-fold proteins in eukaryotic biology.