Complete inhibition of tobamovirus multiplication by simultaneous mutations in two homologous host genes

The TOM1 gene of Arabidopsis thaliana encodes a putative multipass transmembrane protein which is necessary for the efficient multiplication of tobamoviruses. We have previously shown that mutations severely destructive to the TOM1 gene reduce tobamovirus multiplication to low levels but do not impair it completely. In this report, we subjected one of the tom1 mutants (tom1-1) to another round of mutagenesis and isolated a new mutant which did not permit a detectable level of tobamovirus multiplication. In addition to tom1-1, this mutant carried a mutation referred to as tom3-1. Positional cloning showed that TOM3 was one of two TOM1-like genes in Arabidopsis. Based on the similarity between the amino acid sequences of TOM1 and TOM3, together with the results of a Sos recruitment assay suggesting that both TOM1 and TOM3 bind tobamovirus-encoded replication proteins, we propose that TOM1 and TOM3 play parallel and essential roles in the replication of tobamoviruses.     

TOM1 family conservation within the plant kingdom for tobacco mosaic virus accumulation

The susceptibility factor TOBAMOVIRUS MULTIPLICATION 1 (TOM1) is required for efficient multiplication of tobacco mosaic virus (TMV). Although some phylogenetic and functional analyses of the TOM1 family members have been conducted, a comprehensive analysis of the TOM1 homologues based on phylogeny from the most ancient to the youngest representatives within the plant kingdom, analysis of support for tobamovirus accumulation and interaction with other host and viral proteins has not been reported. In this study, using Nicotiana benthamiana and TMV as a model system, we functionally characterized the TOM1 homologues from N. benthamiana and other plant species from different plant lineages. We modified a multiplex genome editing tool and generated a sextuple mutant in which TMV multiplication was dramatically inhibited. We showed that TOM1 homologues from N. benthamiana exhibited variable capacities to support TMV multiplication. Evolutionary analysis revealed that the TOM1 family is restricted to the plant kingdom and probably originated in the Chlorophyta division, suggesting an ancient origin of the TOM1 family. We found that the TOM1 family acquired the ability to promote TMV multiplication after the divergence of moss and spikemoss. Moreover, the capacity of TOM1 orthologues from different plant species to promote TMV multiplication and the interactions between TOM1 and TOM2A and between TOM1 and TMV-encoded replication proteins are highly conserved, suggesting a conserved nature of the TOM2A–TOM1–TMV Hel module in promoting TMV multiplication. Our study not only revealed a conserved nature of a gene module to promote tobamovirus multiplication, but also provides a valuable strategy for TMV-resistant crop development.     

Subcellular localization of host and viral proteins associated with tobamovirus RNA replication

Arabidopsis TOM1 (AtTOM1) and TOM2A (AtTOM2A) are integral membrane proteins genetically identified to be necessary for efficient intracellular multiplication of tobamoviruses. AtTOM1 interacts with the helicase domain polypeptide of tobamovirus-encoded replication proteins and with AtTOM2A, suggesting that both AtTOM1 and AtTOM2A are integral components of the tobamovirus replication complex. We show here that AtTOM1 and AtTOM2A proteins tagged with green fluorescent protein (GFP) are targeted to the vacuolar membrane (tonoplast)-like structures in plant cells. In subcellular fractionation analyses, GFP-AtTOM2A, AtTOM2A and its tobacco homolog NtTOM2A were predominantly fractionated to low-density tonoplast-rich fractions, whereas AtTOM1-GFP, AtTOM1 and its tobacco homolog NtTOM1 were distributed mainly into the tonoplast-rich fractions and partially into higher-buoyant-density fractions containing membranes from several other organelles. The tobamovirus-encoded replication proteins were co-fractionated with both NtTOM1 and viral RNA-dependent RNA polymerase activity. The replication proteins were also found in the fractions containing non-membrane-bound proteins, but neither NtTOM1 nor the polymerase activity was detected there. These observations suggest that the formation of tobamoviral RNA replication complex occurs on TOM1-containing membranes and is facilitated by TOM2A.     

A host small GTP-binding protein ARL8 plays crucial roles in tobamovirus RNA replication

Tomato mosaic virus (ToMV), like other eukaryotic positive-strand RNA viruses, replicates its genomic RNA in replication complexes formed on intracellular membranes. Previous studies showed that a host seven-pass transmembrane protein TOM1 is necessary for efficient ToMV multiplication. Here, we show that a small GTP-binding protein ARL8, along with TOM1, is co-purified with a FLAG epitope-tagged ToMV 180K replication protein from solubilized membranes of ToMV-infected tobacco (Nicotiana tabacum) cells. When solubilized membranes of ToMV-infected tobacco cells that expressed FLAG-tagged ARL8 were subjected to immunopurification with anti-FLAG antibody, ToMV 130K and 180K replication proteins and TOM1 were co-purified and the purified fraction showed RNA-dependent RNA polymerase activity that transcribed ToMV RNA. From uninfected cells, TOM1 co-purified with FLAG-tagged ARL8 less efficiently, suggesting that a complex containing ToMV replication proteins, TOM1, and ARL8 are formed on membranes in infected cells. In Arabidopsis thaliana, ARL8 consists of four family members. Simultaneous mutations in two specific ARL8 genes completely inhibited tobamovirus multiplication. In an in vitro ToMV RNA translation-replication system, the lack of either TOM1 or ARL8 proteins inhibited the production of replicative-form RNA, indicating that TOM1 and ARL8 are required for efficient negative-strand RNA synthesis. When ToMV 130K protein was co-expressed with TOM1 and ARL8 in yeast, RNA 5'-capping activity was detected in the membrane fraction. This activity was undetectable or very weak when the 130K protein was expressed alone or with either TOM1 or ARL8. Taken together, these results suggest that TOM1 and ARL8 are components of ToMV RNA replication complexes and play crucial roles in a process toward activation of the replication proteins' RNA synthesizing and capping functions.     

Tobamovirus-resistant tobacco generated by RNA interference directed against host genes

Two homologous Nicotiana tabacum genes NtTOM1 and NtTOM3 have been identified. These genes encode polypeptides with amino acid sequence similarity to Arabidopsis thaliana TOM1 and TOM3, which function in parallel to support tobamovirus multiplication. Simultaneous RNA interference against NtTOM1 and NtTOM3 in N. tabacum resulted in nearly complete inhibition of the multiplication of Tomato mosaic virus and other tobamoviruses, but did not affect plant growth or the ability of Cucumber mosaic virus to multiply. As TOM1 and TOM3 homologues are present in a variety of plant species, their inhibition via RNA interference should constitute a useful method for generating tobamovirus-resistant plants.     

Role of the Negative Charges in the Cytosolic Domain of TOM22 in the Import of Precursor Proteins into Mitochondria

TOM22 is an essential mitochondrial outer membrane protein required for the import of precursor proteins into the organelles. The amino-terminal 84 amino acids of TOM22 extend into the cytosol and include 19 negatively and 6 positively charged residues. This region of the protein is thought to interact with positively charged presequences on mitochondrial preproteins, presumably via electrostatic interactions. We constructed a series of mutant derivatives of TOM22 in which 2 to 15 of the negatively charged residues in the cytosolic domain were changed to their corresponding amido forms. The mutant constructs were transformed into a sheltered Neurospora crassa heterokaryon bearing a tom22::hygromycin R disruption in one nucleus. All constructs restored viability to the disruption-carrying nucleus and gave rise to homokaryotic strains containing mutant tom22 alleles. Isolated mitochondria from three representative mutant strains, including the mutant carrying 15 neutralized residues (strain 861), imported precursor proteins at efficiencies comparable to those for wild-type organelles. Precursor binding studies with mitochondrial outer membrane vesicles from several of the mutant strains, including strain 861, revealed only slight differences from binding to wild-type vesicles. Deletion mutants lacking portions of the negatively charged region of TOM22 can also restore viability to the disruption-containing nucleus, but mutants lacking the entire region cannot. Taken together, these data suggest that an abundance of negative charges in the cytosolic domain of TOM22 is not essential for the binding or import of mitochondrial precursor proteins; however, other features in the domain are required.     

Effects of the tom1 mutation of Arabidopsis thaliana on the multiplication of tobacco mosaic virus RNA in protoplasts.

For the multiplication of RNA viruses, specific host factors are considered essential, but as of yet little is known about this aspect of virus multiplication. To identify such host factors, we previously isolated PD114, a mutant of Arabidopsis thaliana, in which the accumulation of the coat protein of tobacco mosaic virus (TMV) in uninoculated leaves of an infected plant was reduced to low levels. The causal mutation, designated tom1, was single, nuclear, and recessive. Here, we demonstrate that the tom1 mutation affects the amplification of TMV-related RNAs in a single cell. When protoplasts were inoculated with TMV RNA by electroporation, the percentage of TMV-positive protoplasts (detected by indirect immunofluorescence staining with anti-TMV antibodies) was lower (about 1/5 to 1/10) among PD114 protoplasts than among wild-type protoplasts. In TMV-positive PD114 protoplasts, the amounts of the positive-strand RNAs (the genomic RNA and subgenomic mRNAs) and coat protein reached levels similar to, or slightly lower than, those reached in TMV-positive wild-type protoplasts, but the accumulation of the positive-strand RNAs and coat protein occurred more slowly than with the wild-type protoplasts. The parallel decrease in the amounts of the coat protein and its mRNA suggests that the coat protein is translated from its mRNA with normal efficiency. These observations support the idea that the TOM1 gene encodes a host factor necessary for the efficient amplification of TMV RNA in an infected cell. Furthermore, we show that TMV multiplication in PD114 protoplasts is severely affected by the coinoculation of cucumber mosaic virus (CMV) RNA. When PD114 protoplasts were inoculated with a mixture of TMV and CMV RNAs by electroporation, the accumulation of TMV-related molecules was approximately one-fifth of that in PD114 protoplasts inoculated with TMV RNA alone. No such reduction in the accumulation of TMV-related molecules was observed when wild-type protoplasts were inoculated with a mixture of TMV and CMV RNAs or when wild-type and PD114 protoplasts were inoculated with a mixture of TMV and turnip crinkle virus RNAs. These observations are compatible with a hypothetical model in which a gene(s) that is distinct from the TOM1 gene is involved in both TMV and CMV multiplication.     

Structural requirements of Tom40 for assembly into preexisting TOM complexes of mitochondria

Tom40 is the major subunit of the translocase of the outer mitochondrial membrane (the TOM complex). To study the assembly pathway of Tom40, we have followed the integration of the protein into the TOM complex in vitro and in vivo using wild-type and altered versions of the Neurospora crassa Tom40 protein. Upon import into isolated mitochondria, Tom40 precursor proteins lacking the first 20 or the first 40 amino acid residues were assembled as the wild-type protein. In contrast, a Tom40 precursor lacking residues 41 to 60, which contains a highly conserved region of the protein, was arrested at an intermediate stage of assembly. We constructed mutant versions of Tom40 affecting this region and transformed the genes into a sheltered heterokaryon containing a tom40 null nucleus. Homokaryotic strains expressing the mutant Tom40 proteins had growth rate defects and were deficient in their ability to form conidia. Analysis of the TOM complex in these strains by blue native gel electrophoresis revealed alterations in electrophoretic mobility and a tendency to lose Tom40 subunits from the complex. Thus, both in vitro and in vivo studies implicate residues 41 to 60 as containing a sequence required for proper assembly/stability of Tom40 into the TOM complex. Finally, we found that TOM complexes in the mitochondrial outer membrane were capable of exchanging subunits in vitro. A model is proposed for the integration of Tom40 subunits into the TOM complex.     

Effect of mutations in Tom40 on stability of the translocase of the outer mitochondrial membrane (TOM) complex, assembly of Tom40, and import of mitochondrial preproteins

Mitochondrial preproteins synthesized in the cytosol are imported through the mitochondrial outer membrane by the translocase of the outer mitochondrial membrane (TOM) complex. Tom40 is the major component of the complex and is essential for cell viability. We generated 21 different mutations in conserved regions of the Neurospora crassa Tom40 protein. The mutant genes were transformed into a tom40 null nucleus maintained in a sheltered heterokaryon, and 17 of the mutant genes gave rise to viable strains. All mutations reduced the efficiency of the altered Tom40 molecules to assemble into the TOM complex. Mitochondria isolated from seven of the mutant strains had defects for importing mitochondrial preproteins. Only one strain had a general import defect for all preproteins examined. Another mutation resulted in defects in the import of a matrix-destined preprotein and an outer membrane beta-barrel protein, but import of the ADP/ATP carrier to the inner membrane was unaffected. Five strains showed deficiencies in the import of beta-barrel proteins. The latter results suggest that the TOM complex distinguishes beta-barrel proteins from other classes of preprotein during import. This supports the idea that the TOM complex plays an active role in the transfer of preproteins to subsequent translocases for insertion into the correct mitochondrial subcompartment.     

Yeast Pdr13p and Zuo1p molecular chaperones are new functional Hsp70 and Hsp40 partners

The deletion of the TOM1 gene encoding a putative ubiquitin ligase causes a temperature sensitive cellular growth in Saccharomyces cerevisiae. The arrested cells exhibit pleiotropic defects in nuclear division, maintenance of nuclear structure and heat stress responses. We previously identified a zuo1 mutation as an extragenic suppressor of the tom1 mutant. ZUO1 encodes a DnaJ-related Hsp40. Here we show that a recessive cold sensitive mutation in PDR13 coding for an Hsp70 suppressed the tom1 mutation. The pdr13 deletion mutant was sensitive to high osmolarity, just like the zuo1 deletion mutant. A zuo1 pdr13 double deletion mutant did not show additive phenotypes. Furthermore, a tagged-Zuo1p was co-immunoprecipitated with a tagged-Pdr13p. Taken together, we propose that Pdr13p and Zuo1p are a new pair of Hsp70:Hsp40 molecular chaperones. In addition, Pdr13p co-sedimented with translating ribosomes and this association was independent of the presence of Zuo1p.     

Genes encoding ribosomal proteins Rps0A/B of Saccharomyces cerevisiae interact with TOM1 mutants defective in ribosome synthesis

The Saccharomyces cerevisiae RPS0A/B genes encode proteins of the 40S ribosomal subunit that are required for the maturation of 18S rRNA. We show here that the RPS0 genes interact genetically with TOM1. TOM1 encodes a member of the hect-domain-containing E3 ubiquitin-protein ligase family that is required for growth at elevated temperatures. Mutant alleles of the RPS0 and TOM1 genes have synergistic effects on cell growth at temperatures permissive for TOM1 mutants. Moreover, the growth arrest of TOM1 mutants at elevated temperatures is partially suppressed by overexpression of RPS0A/B. Strains with mutant alleles of TOM1 are defective in multiple steps in rRNA processing, and interactions between RPS0A/B and TOM1 stem, in part, from their roles in the maturation of ribosomal subunits. Ribosome synthesis is therefore included among the cellular processes governed by members of the hect-domain-containing E3 ubiquitin-protein ligase family.     

An unusual TOM20/TOM22 bypass mechanism for the mitochondrial targeting of cytochrome P450 proteins containing N-terminal chimeric signals

Previously we showed that xenobiotic-inducible cytochrome P450 (CYP) proteins are bimodally targeted to the endoplasmic reticulum and mitochondria. In the present study, we investigated the mechanism of delivery of chimeric signal-containing CYP proteins to the peripheral and channel-forming mitochondrial outer membrane translocases (TOMs). CYP+33/1A1 and CYP2B1 did not require peripheral TOM70, TOM20, or TOM22 for translocation through the channel-forming TOM40 protein. In contrast, CYP+5/1A1 and CYP2E1 were able to bypass TOM20 and TOM22 but required TOM70. CYP27, which contains a canonical cleavable mitochondrial signal, required all of the peripheral TOMs for its mitochondrial translocation. We investigated the underlying mechanisms of bypass of peripheral TOMs by CYPs with chimeric signals. The results suggested that interaction of CYPs with Hsp70, a cytosolic chaperone involved in the mitochondrial import, alone was sufficient for the recognition of chimeric signals by peripheral TOMs. However, sequential interaction of chimeric signal-containing CYPs with Hsp70 and Hsp90 resulted in the bypass of peripheral TOMs, whereas CYP27 interacted only with Hsp70 and was not able to bypass peripheral TOMs. Our results also show that delivery of chimeric signal-containing client proteins by Hsp90 required the cytosol-exposed N-terminal 143 amino acids of TOM40. TOM40 devoid of this domain was unable to bind CYP proteins. These results suggest that, compared with the unimodal mitochondria-targeting signals, the chimeric mitochondria-targeting signals are highly evolved and dynamic in nature.     

Identification of mammalian TOM22 as a subunit of the preprotein translocase of the mitochondrial outer membrane

A mitochondrial outer membrane protein of approximately 22 kDa (1C9-2) was purified from Vero cells assessing immunoreactivity with a monoclonal antibody, and the cDNA was cloned based on the partial amino acid sequence of the trypsin-digested fragments. 1C9-2 had 19-20% sequence identity to fungal Tom22, a component of the preprotein translocase of the outer membrane (the TOM complex) with receptor and organizer functions. Despite such a low sequence identity, both shared a remarkable structural similarity in the hydrophobicity profile, membrane topology in the Ncyt-Cin orientation through a transmembrane domain in the middle of the molecule, and the abundant acidic amino acid residues in the N-terminal domain. The antibodies against 1C9-2 inhibited the import of a matrix-targeted preprotein into isolated mitochondria. Blue native polyacrylamide gel electrophoresis of digitonin-solubilized outer membranes revealed that 1C9-2 is firmly associated with TOM40 in the approximately 400-kDa complex, with a size and composition similar to those of the fungal TOM core complex. Furthermore, 1C9-2 complemented the defects of growth and mitochondrial protein import in Deltatom22 yeast cells. Taken together, these results demonstrate that 1C9-2 is a functional homologue of fungal Tom22 and functions as a component of the TOM complex.     

Endofin recruits TOM1 to endosomes

Endofin is an endosomal protein implicated in regulating membrane trafficking. It is characterized by the presence of a phosphatidylinositol 3-phosphate-binding FYVE domain positioned in the middle of the molecule. To determine its potential effectors or binding partners, we used the carboxyl-terminal half of endofin as bait to screen a human brain cDNA library in a yeast two-hybrid system. Three clones that encode TOM1 were recovered. TOM1 is a protein closely related to the VHS (VPS-27, Hrs, and STAM) domain-containing GGA family. Although the function of the GGAs in mediating Golgi to lysosomal trafficking is well established, the subcellular localization and function of TOM1 remain unknown. Glutathione S-transferase pull-down assays as well as co-immunoprecipitation experiments confirmed that the carboxyl-terminal half of endofin binds specifically to the carboxyl-terminal region of TOM1. Neither SARA nor Hrs, two other FYVE domain proteins, interact with this region of TOM1. Moreover, endofin does not interact with the analogous region of two other members of the TOM1 protein family, namely, TOM1-like 1 (TOM1-L1) or TOM1-like 2 (TOM1-L2). The carboxyl-terminal region of TOM1 was used as immunogen to generate TOM1-specific antibody. This antibody can distinguish TOM1 from the other family members as well as coimmunoprecipitate endogenous endofin. It also revealed the primarily cytosolic distribution of TOM1 in a variety of cell types by immunofluorescence analyses. In addition, sucrose density gradient analysis showed that both TOM1 and endofin can be detected in cellular compartments marked by the early endosomal marker EEA1. A marked recruitment of TOM1 to endosomes was observed in cells overexpressing endofin or its carboxyl-terminal fragment, indicating TOM1 to be an effector for endofin and suggesting a possible role for TOM1 in endosomal trafficking.     

The phenotype of the minichromosome maintenance mutant mcm3 is characteristic of mutants defective in DNA replication.

MCM3 is an essential gene involved in the maintenance of minichromosomes in yeast cells. It encodes a protein of 971 amino acids that shows striking homology to the Mcm2 protein. We have mapped the mcm3-1 mutation of the left arm of chromosome V approximately 3 kb centromere proximal of anp1. The mcm3-1 mutant was found to be thermosensitive for growth. Under permissive growth conditions, it was defective in minichromosome maintenance in an autonomously replicating sequence-specific manner and showed an increase in chromosome loss and recombination. Under nonpermissive conditions, mcm3-1 exhibited a cell cycle arrest phenotype, arresting at the large-bud stage with an undivided nucleus that had a DNA content of nearly 2n. These phenotypes are consistent with incomplete replication of the genome of the mcm3-1 mutant, possibly as a result of limited replication initiation at selective autonomously replicating sequences leading to cell cycle arrest before mitosis. The phenotype exhibited by the mcm3 mutant is very similar to that of mcm2, suggesting that the Mcm2 and Mcm3 protein may play interacting roles in DNA replication.     

Yeast tom1 mutant exhibits pleiotropic defects in nuclear division, maintenance of nuclear structure and nucleocytoplasmic transport at high temperatures.

A tom1-1 mutant was isolated from Saccharomyces cerevisiae. At high temperatures, 60% of the cells were arrested as dumbbell forms with a single large nucleus containing duplicated DNA and a short spindle. Electron-microscopy showed electron-dense structures scattered within the nucleus. Indirect immunofluorescent microscopy revealed these structures to be fragmented nucleoli since the dotted structures were stained with anti-Nop1(fibrillarin) antibody in large regions of the nuclei. Fluorescent in situ hybridization analysis using oligo(dT) revealed nuclear accumulation of poly(A)+RNA. We cloned TOM1 which encodes a large protein (380kDa) with a hect (homologous to E6-AP C terminus)-domain at its C terminus. Deletions of either this hect-region or the entire gene made cellular growth temperature-sensitive. Site-directed mutagenesis of the conserved cysteine residue (tom1C3235A) in the hect-domain, supposed to be necessary for thioester-bond formation with ubiquitin, abolished the gene function. When a functional glutathione S-transferase (GST)-tagged hect protein was overproduced, it facilitated the protein conjugation with a myc-tagged ubiquitinRA, while this was not seen when GST-hectC3235A was overproduced. The protein conjugation with a hemagglutinin-tagged Smt3 was not affected by the overproduction of GST-hect. Taken together, we suggest that Tom1 is a ubiquitin ligase. As a multi-copy suppressor of tom1, we isolated STM3/NPI46/FPR3 which encodes a nucleolar nucleolin-like protein. We discuss possible functions of Tom1 with respect to the pleiotropic defects of nuclear division, maintenance of nuclear structure, and nucleocytoplasmic transport.     

Characterization of Neurospora crassa Tom40-deficient mutants and effect of specific mutations on Tom40 assembly

The TOM complex (Translocase of the Outer mitochondrial Membrane) is responsible for the recognition of mitochondrial preproteins synthesized in the cytosol and for their translocation across or into the outer mitochondrial membrane. Tom40 is the major component of the TOM complex and forms the translocation pore. We have created a tom40 mutant of Neurospora crassa and have demonstrated that the gene is essential for the viability of the organism. Mitochondria with reduced levels of Tom40 were deficient for import of mitochondrial preproteins and contained reduced levels of the TOM complex components Tom22 and Tom6, suggesting that the import and/or stability of these proteins is dependent on the presence of Tom40. Mutant Tom40 preproteins were analyzed for their ability to be assembled into the TOM complex. In vitro import assays revealed that conserved regions near the N terminus (residues 51-60) and the C terminus (residues 321-323) of the 349-amino acid protein were required for assembly beyond a 250-kDa intermediate form. Mutant strains expressing Tom40 with residues 51-60 deleted were viable but exhibited growth defects. Slow growing mutants expressing Tom40, where residues 321-323 were changed to Ala residues, were isolated but showed TOM complex defects, whereas strains in which residues 321-323 were deleted could not be isolated. Analysis of the assembly of mutant Tom40 precursors in vitro supported a previous model in which Tom40 precursors progress from the 250-kDa intermediate to a 100-kDa form and then assemble into the 400-kDa TOM complex. Surprisingly, when wild type mitochondria containing Tom40 precursors arrested at the 250-kDa intermediate were treated with sodium carbonate, further assembly of intermediates into the TOM complex occurred, suggesting that disruption of protein-protein interactions may facilitate assembly. Import of wild type Tom40 precursor into mitochondria containing a mutant Tom40 lacking residues 40-48 revealed an alternate assembly pathway and demonstrated that the N-terminal region of pre-existing Tom40 molecules in the TOM complex plays a role in the assembly of incoming Tom40 molecules.