吉久 徹

The Saccharomyces cerevisiae uses a highly glycolytic metabolism, if glucose is available, through appropriately suppressing mitochondrial functions except for some of them such as Fe/S cluster biogenesis. Puf3p, a Pumillio family protein, plays a pivotal role in modulating mitochondrial activity, especially during fermentation, by destabilizing its target mRNAs and/or by repressing their translation. Puf3p preferentially binds to 8-nt conserved binding sequences in the 3′-UTR of nuclear-encoded mitochondrial (nc-mitochondrial) mRNAs, leading to broad effects on gene expression under fermentable conditions. To further explore how Puf3p post-transcriptionally regulates nc-mitochondrial mRNAs in response to cell growth conditions, we initially focused on nc-mitochondrial mRNAs known to be enriched in monosomes in a glucose-rich environment. We unexpectedly found that one of the monosome-enriched mRNAs, CAT5/COQ7 mRNA, directly interacts with Puf3p through its non-canonical Puf3p binding sequence, which is generally less considered as a Puf3p binding site. Western blot analysis showed that Puf3p represses translation of Cat5p, regardless of culture in fermentable or respiratory medium. In vitro binding assay confirmed Puf3p’s direct interaction with CAT5 mRNA via this non-canonical Puf3p-binding site. Although cat5 mutants of the non-canonical Puf3p-binding site grow normally, Cat5p expression is altered, indicating that CAT5 mRNA is a bona fide Puf3p target with additional regulatory factors acting through this sequence. Unlike other yeast PUF proteins, Puf3p uniquely regulates Cat5p by destabilizing mRNA and repressing translation, shedding new light on an unknown part of the Puf3p regulatory network. Given that pathological variants of human COQ7 lead to CoQ₁₀ deficiency and yeast cat5Δ can be complemented by hCOQ7, our findings may also offer some insights into clinical aspects of COQ7-related disorders.

Saccharomyces cerevisiae has six synonymous tRNATrpCCA genes encoding the identical sequence, including their intronic region. They are supposed to express tRNATrpCCA in the same quality and quantity. Here, we generated single to quintuple deletion strains with all the possible combinations of the synonymous tRNATrpCCA genes to analyze whether those individual genes equally contribute cell viability and tRNA production. The quintuple deletion strains that only harbor tW(CCA)J, tW(CCA)M, or tW(CCA)P were viable but almost lethal while the other quintuple deletions showed moderately impaired growth. Theses growth differences were not obvious among the quadruple deletion strains, which expressed almost one third of mature tRNATrpCCA in the wild type. Therefore, no dosage compensation operates for tRNATrpCCA amount, and growth variations among the quintuple deletion strains may not simply reflect differences in tRNATrpCCA shortage. Yeast may retain the redundancy of tRNATrpCCA genes for a noncanonical function(s) beyond supply of the tRNA to translation.

ABSTRACT Yeasts generally grow with a highly glycolytic metabolism and restrain mitochondrial biogenesis except for some Fe/S proteins. Respiratory mitochondrial functions and biosynthesis pathways are well studied, however how cells coordinate basal fermentative mitochondrial functions is not fully understood. We show that a part of nuclear-encoded mitochondrial mRNAs, which preferentially translated in monosomes, are regulated by Puf3p upon glucose-rich media. Especially, of those monosome-enriched nuclear-encoded mitochondrial mRNAs, CAT5/COQ7 mRNA has a variant of the canonical Puf3p binding site on its 3’-UTR. Western blot analysis showed that Puf3p represses the translation of Cat5p regardless of fermentable or respiratory media. In vitro binding assay revealed that Puf3p directly binds to CAT5 mRNA via the non-canonical Puf3p binding site. Mutants harboring the substitution of the non-canonical Puf3p binding site in CAT5 mRNA grew normally but impaired Cat5p expressions apparently, indicating CAT5 mRNA is a bona fide Puf3p target. Overall, Puf3p, a general key modulator for nuclear-encoded mitochondrial mRNAs, fine-tunes translation of a subset of nuclear-encoded mitochondrial mRNAs including mRNAs with non-canonical Puf3p binding sites under the fermentation. This may be required to keeping the fundamental functions of yeast mitochondria at proper levels.

To maintain optimal proteome, both codon choice of each mRNA and supply of aminoacyl-tRNAs are two principal factors in translation. Recent reports have revealed that the amounts of tRNAs in cells are more dynamic than we had expected. High-throughput methods such as RNA-Seq and microarrays are versatile for comprehensive detection of changes in individual tRNA amounts, but they suffer from inability to assess signal production efficiencies of individual tRNA species. Thus, they are not the perfect choice to measure absolute amounts of tRNAs. Here, we introduce a novel method for this purpose, termed Oligonucleotide-directed Three-prime Terminal Extension of RNA (OTTER), which employs fluorescence-labeling at the 3'-terminus of a tRNA by optimized reverse primer extension and an assessment step of each labeling efficiency by northern blotting. Using this method, we quantified the absolute amounts of the 34 individual and 4 pairs of isoacceptor tRNAs out of the total 42 nuclear-encoded isoacceptors in the yeast Saccharomyces cerevisiae. We found that the amounts of tRNAs in log phase yeast cells grown in a rich glucose medium range from 0.030 to 0.73 pmol/µg RNA. The tRNA amounts seem to be altered at the isoacceptor level by a few folds in response to physiological growing conditions. The data obtained by OTTER are poorly correlated with those by simple RNA-Seq, marginally with those by microarrays and by microscale thermophoresis. However, the OTTER data showed good agreement with the data obtained by 2D-gel analysis of in vivo radiolabeled RNAs. Thus, OTTER is a suitable method for quantifying absolute amounts of tRNAs at the level of isoacceptor resolution.

<title>Abstract</title>eIF2α phosphorylation-mediated translational regulation is crucial for global translation repression by various stresses, including the unfolded protein response (UPR). However, translational control during UPR has not been demonstrated in yeast. This study investigated ribosome ubiquitination-mediated translational controls during UPR. Tunicamycin-induced ER stress enhanced the levels of ubiquitination of the ribosomal proteins uS10, uS3 and eS7. Not4-mediated monoubiquitination of eS7A was required for resistance to tunicamycin, whereas E3 ligase Hel2-mediated ubiquitination of uS10 was not. Ribosome profiling showed that the monoubiquitination of eS7A was crucial for translational regulation, including the upregulation of the spliced form of HAC1 (<italic>HAC1i</italic>) mRNA and the downregulation of Histidine triad NucleoTide-binding 1 (<italic>HNT1</italic>) mRNA. Downregulation of the deubiquitinating enzyme complex Upb3-Bre5 increased the levels of ubiquitinated eS7A during UPR in an Ire1-independent manner. These findings suggest that the monoubiquitination of ribosomal protein eS7A plays a crucial role in translational controls during the ER stress response in yeast.

In eukaryotes and archaea, tRNA genes frequently contain introns, which are removed during maturation. However, biological roles of tRNA introns remain elusive. Here, we constructed a complete set of Saccharomyces cerevisiae strains in which the introns were removed from all the synonymous genes encoding 10 different tRNA species. All the intronless strains were viable, but the tRNAPheGAA and tRNATyrGUA intronless strains displayed slow growth, cold sensitivity and defective growth under respiratory conditions, indicating physiological importance of certain tRNA introns. Northern analyses revealed that removal of the introns from genes encoding three tRNAs reduced the amounts of the corresponding mature tRNAs, while it did not affect aminoacylation. Unexpectedly, the tRNALeuCAA intronless strain showed reduced 5.8S rRNA levels and abnormal nucleolar morphology. Because pseudouridine (Ψ) occurs at position 34 of the tRNAIleUAU anticodon in an intron-dependent manner, tRNAIleUAU in the intronless strain lost Ψ34. However, in a portion of tRNAIleUAU population, position 34 was converted into 5-carbamoylmethyluridine (ncm5U), which could reduce decoding fidelity. In summary, our results demonstrate that, while introns are dispensable for cell viability, some introns have diverse roles, such as ensuring proper growth under various conditions and controlling the appropriate anticodon modifications for accurate pairing with the codon.

tRNAs, a class of non-coding RNAs essential for translation, are unique among cytosolic RNA species in that they shuttle between the nucleus and cytoplasm during their life. Although their export from the nucleus has been studied in detail, limited information on import machinery was available. Our group recently reported that Ssa2p, one of major cytosolic Hsp70s in Saccharomyces cerevisiae, acts as a crucial factor for tRNA import upon nutrient starvation. Ssa2p can bind tRNAs and a nucleoporin directly in an ATP-sensitive manner, suggesting that it acts as a nuclear import carrier for tRNAs, like importin- proteins. In vitro assays revealed that Ssa2p binds tRNA specifically but has preference for loosely folded tRNAs. In this Extra View, these features of Ssa2p as a new import factor is discussed with other recent findings related to nucleocytoplasmic transport of tRNAs reported from other groups.

Background: The yeast tRNA splicing endonuclease (Sen) complex is located on the mitochondrial outer membrane and splices precursor tRNAs. Results: The Sen complex cleaves the mitochondria-localized CBP1 mRNA in vivo and in vitro. Conclusion: Mitochondrial localization of the Sen complex is required for the cleavage of the CBP1 mRNA. Significance: This study shows a novel role of the tRNA splicing endonuclease complex in the cleavage of mitochondria-localized mRNA. The tRNA splicing endonuclease (Sen) complex is located on the mitochondrial outer membrane and splices precursor tRNAs in Saccharomyces cerevisiae. Here, we demonstrate that the Sen complex cleaves the mitochondria-localized mRNA encoding Cbp1 (cytochrome b mRNA processing 1). Endonucleolytic cleavage of this mRNA required two cis-elements: the mitochondrial targeting signal and the stem-loop 652-726-nt region. Mitochondrial localization of the Sen complex was required for cleavage of the CBP1 mRNA, and the Sen complex cleaved this mRNA directly in vitro. We propose that the Sen complex cleaves the CBP1 mRNA, which is co-translationally localized to mitochondria via its mitochondrial targeting signal.

tRNAs are unique among various RNAs in that they shuttle between the nucleus and the cytoplasm, and their localization is regulated by nutrient conditions. Although nuclear export of tRNAs has been well documented, the import machinery is poorly understood. Here, we identified Ssa2p, a major cytoplasmic Hsp70 in Saccharomyces cerevisiae, as a tRNA-binding protein whose deletion compromises nuclear accumulation of tRNAs upon nutrient starvation. Ssa2p recognizes several structural features of tRNAs through its nucleotide-binding domain, but prefers loosely-folded tRNAs, suggesting that Ssa2p has a chaperone-like activity for RNAs. Ssa2p also binds Nup116, one of the yeast nucleoporins. Sis1p and Ydj1p, cytoplasmic co-chaperones for Ssa proteins, were also found to contribute to the tRNA import. These results unveil a novel function of the Ssa2p system as a tRNA carrier for nuclear import by a novel mode of substrate recognition. Such Ssa2p-mediated tRNA import likely contributes to quality control of cytosolic tRNAs.

lntrons are found in various tRNA genes in all the three kingdoms of life. Especially, archaeal and eukaryotic genomes are good sources of tRNA introns that are removed by proteinaceous splicing machinery. Most intron-containing tRNA genes both in archaea and eukaryotes possess an intron at a so-called canonical position, one nucleotide 3' to their anticodon, while recent bioinformatics have revealed unusual types of tRNA introns and their derivatives especially in archaeal genomes. Gain and loss of tRNA introns during various stages of evolution are obvious both in archaea and eukaryotes from analyses of comparative genomics. The splicing of tRNA molecules has been studied extensively from biochemical and cell biological points of view, and such analyses of eukaryotic systems provided interesting findings in the past years. Here, I summarize recent progresses in the analyses of tRNA introns and the splicing process, and try to clarify new and old questions to be solved in the next stages.

The membrane topology of Om45 in the yeast mitochondrial outer membrane ( OM) is under debate. Here, we confirm that Om45 is anchored to the OM from the intermembrane space (IMS) by its N-terminal hydrophobic segment. We show that import of Om45 requires the presequence receptors, Tom20 and Tom22, and the import channel of Tom40. Unlike any of the known OM proteins, Om45 import requires the TIM23 complex in the inner membrane, a translocator for presequence-containing proteins, and the membrane potential (Delta Psi). Therefore, Om45 is anchored to the OM via the IMS by a novel import pathway involving the TIM23 complex.

In all eukaryotes, transcribed precursor tRNAs are maturated by processing and modification processes in nucleus and are transported to the cytoplasm. The cytoplasmic export protein (Cex1p) captures mature tRNAs from the nuclear export receptor (Los1p) on the cytoplasmic side of the nuclear pore complex, and it delivers them to eukaryotic elongation factor 1 alpha. This conserved Cex1p function is essential for the quality control of mature tRNAs to ensure accurate translation. However, the structural basis of how Cex1p recognizes tRNAs and shuttles them to the translational apparatus remains unclear. Here, we solved the 2.2 angstrom resolution crystal structure of Saccharomyces cerevisiae Cex1p with C-terminal 197 disordered residues truncated. Cex1p adopts an elongated architecture, consisting of N-terminal kinase-like and a C-terminal alpha-helical HEAT repeat domains. Structure-based biochemical analyses suggested that Cex1p binds tRNAs on its inner side, using the positively charged HEAT repeat surface and the C-terminal disordered region. The N-terminal kinase-like domain acts as a scaffold to interact with the Ran-exportin (Los1p center dot Gsp1p) machinery. These results provide the structural basis of Los1p center dot Gsp1p center dot Cex1p center dot tRNA complex formation, thus clarifying the dynamic mechanism of tRNA shuttling from exportin to the translational apparatus.

A part of eukaryotic tRNA genes harbor an intron at one nucleotide 3&apos; to the anticodon, so that removal of the intron is an essential processing step for tRNA maturation. While some tRNA introns have important roles in modification of certain nucleotides, essentiality of the tRNA intron in eukaryotes has not been tested extensively. This is partly because most of the eukaryotic genomes have multiple genes encoding an isoacceptor tRNA. Here, we examined whether the intron of tRNA-Trp(CCA) genes, six copies of which are scattered on the genome of yeast, Saccharomyces cerevisiae, is essential for growth or translation of the yeast in vivo. We devised a procedure to remove all of the tRNA introns from the yeast genome iteratively with marker cassettes containing both positive and negative markers. Using this procedure, we removed all the introns from the six tRNA-Trp(CCA) genes, and found that the intronless strain grew normally and expressed tRNA-Trp(CCA) in an amount similar to that of the wild-type genes. Neither incorporation of S-35-labeled amino acids into a TCA-insoluble fraction nor the major protein pattern on SDS-PAGE/2D gel were affected by complete removal of the intron, while expression levels of some proteins were marginally affected. Therefore, the tRNA-Trp(CCA) intron is dispensable for growth and bulk translation of the yeast. This raises the possibility that some mechanism other than selective pressure from translational efficiency maintains the tRNA intron on the yeast genome.

The unfolded protein response (UPR) is an essential signal transduction to cope with protein-folding stress in the endoplasmic reticulum. In the yeast UPR, the unconventional splicing of HAC1 mRNA is a key step. Translation of HAC1 pre-mRNA (HAC1(u) mRNA) is attenuated on polysomes and restarted only after splicing upon the UPR. However, the precise mechanism of this restart remained unclear. Here we show that yeast tRNA ligase (Rlg1p/Trl1p) acting on HAC1 ligation has an unexpected role in HAC1 translation. An RLG1 homologue from Arabidopsis thaliana (AtRLG1) substitutes for yeast RLG1 in tRNA splicing but not in the UPR. Surprisingly, AtRlg1p ligates HAC1 exons, but the spliced mRNA (HAC1(i) mRNA) is not translated efficiently. In the AtRLG1 cells, the HAC1 intron is circularized after splicing and remains associated on polysomes, impairing relief of the translational repression of HAC1(i) mRNA. Furthermore, the HAC1 5&apos; UTR itself enables yeast Rlg1p to regulate translation of the following ORF. RNA IP revealed that yeast Rlg1p is integrated in HAC1 mRNP, before Ire1p cleaves HAC1(u) mRNA. These results indicate that the splicing and the release of translational attenuation of HAC1 mRNA are separable steps and that Rlg1p has pivotal roles in both of these steps.

Mitochondrial protein traffic requires precise recognition of the mitochondrial targeting signals by the import receptors on the mitochondrial surface including a general import receptor Tom20 and a receptor for presequence-less proteins, Tom70. Here we took a proteome-wide approach of mitochondrial protein import in vitro to find a set of presequence-containing precursor proteins for recognition by Tom70. The presequences of the Tom70-dependent precursor proteins were recognized by Tom20, whereas their mature parts exhibited Tom70-dependent import when attached to the presequence of Tom70-independent precursor proteins. The mature parts of the Tom70-dependent precursor proteins have the propensity to aggregate, and the presence of the receptor domain of Tom70 prevents their aggregate formation. Therefore Tom70 plays the role of a docking site for not only cytosolic chaperones but also aggregate-prone substrates to maintain their solubility for efficient transfer to downstream components of the mitochondrial import machineries.

The splicing of nuclear encoded RNAs, including tRNAs, has been widely believed to occur in the nucleus. However, we recently found that one of the tRNA splicing enzymes, splicing endonuclease, is localized to the outer surface of mitochondria in Saccharomyces cerevisiae. These results suggested the unexpected possibility of tRNA splicing in the cytoplasm. To investigate this possibility, we examined whether cytoplasmic pre-tRNAs are bona fide intermediates for tRNA maturation in vivo. We isolated a new reversible allele of temperature-sensitive (ts) sen2 (HA-sen2-42), which encodes a mutant form of one of the catalytic subunits of yeast splicing endonuclease. The HA-sen2-42 cells accumulated large amounts of pre-tRNAs in the cytoplasm at a restrictive temperature, but the pre-tRNAs were diminished when the cells were transferred to a permissive temperature. Using pulse-chase/hybrid-precipitation techniques, we showed that the pre-tRNAs were not degraded but rather converted into mature tRNAs during incubation at the permissive temperature. These and other results indicate that, in S. cerevisiae, pre-tRNAs in the cytoplasm are genuine substrates for splicing, and that the splicing is indeed carried out in the cytoplasm.

The nuclear envelope divides a eukaryotic cell into two compartments, the nucleus and the cytoplasm. Transcription and maturation of RNAs encoded on nuclear chromosomes are carried out in the nucleus, while the proteins coded by these RNAs are translated and processed in the cytoplasm. Cytosolic tRNAs, essential factors for translation, are transcribed in the nucleus and undergo extensive processing before reaching functionality It had previously been believed that tRNAs have only one-way tickets to pass through the nuclear envelope after maturation in the nucleus, and that the small amounts of mature tRNAs found in the nucleus are biosynthetic intermediates. However, two reports from our lab and Anita Hopper's group recently demonstrated that tRNAs have multi-round commuter tickets to shuttle between the nucleus and the cytoplasm in the yeast Saccharomyces cerevisiae. In fact, various tRNA species, including aminoacylated full-length tRNAs and 3' end-shortened tRNAs, are actively imported into the nucleus under various conditions. These findings force a reconsideration of our view of intracellular dynamics of tRNAs, and re-evaluations of the physiological meanings of the nuclear mature tRNAs.

Previous evidence suggested that transfer RNAs (tRNAs) cross the nuclear envelope to the cytosol only once after maturing in the nucleus. We now present evidence for nuclear import of tRNAs in yeast. Several export mutants accumulate mature tRNAs in the nucleus even in the absence of transcription. Import requires energy but not the Ran cycle. These results indicate that tRNAs shuttle between the nucleus and cytosol.

Pre-tRNA splicing has been believed to occur in the nucleus. In yeast, the tRNA splicing endonuclease that cleaves the exon-intron junctions of pre-tRNAs consists of Sen54p, Sen2p, Sen34p, and Sen15p and was thought to be an integral membrane protein of the inner nuclear envelope. Here we show that the majority of Sen2p, Sen54p, and the endonuclease activity are not localized in the nucleus, but on the mitochondrial surface. The endonuclease is peripherally associated with the cytosolic surface of the outer mitochondrial membrane. A Sen54p derivative artificially fixed on the mitochondria as an integral membrane protein can functionally replace the authentic Sen54p, whereas mutant proteins defective in mitochondrial localization are not fully active. sen2 mutant cells accumulate unspliced pre-tRNAs in the cytosol under the restrictive conditions, and this export of the pre-tRNAs partly depends on Los1p, yeast exportin-t. It is difficult to explain these results from the view of tRNA splicing in the nucleus. We rather propose a new possibility that tRNA splicing occurs on the mitochondrial surface in yeast.

The Sec23p/Sec24p complex functions as a component of the COPII coat in vesicle transport from the endoplasmic reticulum. Here we characterize Saccharomyces cerevisiae SEC24, which encodes a protein of 926 amino acids (YIL109C), and a close homologue, ISS1 (YNL049C), which is 55% identical to SEC24. SEC24 is essential for vesicular transport in vivo because depletion of Sec24p is lethal, causing exaggeration of the endoplasmic reticulum and a block in the maturation of carboxypeptidase Y. Overproduction of Sec24p suppressed the temperature sensitivity of sec23-2, and overproduction of both Sec24p and Sec23p suppressed the temperature sensitivity of sec16-2. SEC24 gene disruption could be complemented by overexpression of ISS1, indicating functional redundancy between the two homologous proteins. Deletion of ISS1 had no significant effect on growth or secretion; however, iss1 Delta mutants were found to be synthetically lethal with mutations in the v-SNARE genes SEC22 and BET1. Moreover, overexpression of ISS1 could suppress mutations in SEC22. These genetic interactions suggest that Iss1p may be specialized for the packaging or the function of COPII v-SNAREs. Iss1p tagged with His(6) at its C terminus copurified with Sec23p. Pure Sec23p/Iss1p could replace Sec23p/ Sec24p in the packaging of a soluble cargo molecule (alpha-factor) and v-SNAREs (Sec22p and Bet1p) into COPII vesicles. Abundant proteins in the purified vesicles produced with Sec23p/Iss1p were indistinguishable from those in the regular COPII vesicles produced with Sec23p/Sec24p.

SecA, the preprotein-driving ATPase in Escherichia coli, was shown previously to insert deeply into the plasma membrane in the presence of ATP and a preprotein; this movement of SecA was proposed to be mechanistically coupled with preprotein translocation. We now address the role played by SecY, the central subunit of the membrane-embedded heterotrimeric complex, in the SecA insertion reaction. We identified a secY mutation (secY205), affecting the most carboxy-terminal cytoplasmic domain, that did not allow ATP and preprotein-dependent productive SecA insertion. while allowing idling insertion without the preprotein. Thus, the secY205 mutation might affect the SecYEG 'channel' structure in accepting the preprotein-SecA complex or its opening by the complex. We isolated secA mutations that allele-specifically suppressed the secY205 translocation defect in vivo. One mutant protein, SecA36, with an amino acid alteration near the high-affinity ATP-binding site, was purified and suppressed the in vitro translocation defect of the inverted membrane vesicles carrying the SecY205 protein. The SecA36 protein could also insert into the mutant membrane vesicles in vitro. These results provide genetic evidence that SecA and SecY specifically interact, and show that SecY plays an essential role in insertion of SecA in response to a preprotein and ATP and suggest that SecA drives protein translocation by inserting into the membrane in vivo.

Protein translocation across the plasma membrane of E coli is facilitated by Sec factors, including the membrane-embedded SecYEG subunit and the SecA ATPase. Although there is complete agreement that SecA is essential for protein translocation, some publications question the essentialness of SecY. We previously isolated a number of cold-sensitive mutants of secY and characterized their in vivo phenotypes. In this study, we characterized membrane vesicles prepared from these mutants with respect to their in vitro activities to support protein translocation and to activate the SecA ATPase. These studies revealed several single amino acid alterations that abolish these in vitro activities of membrane vesicles. In particular, several mutations in the two most carboxy-terminal cytoplasmic domains of SecY prevented SecA from functioning as the translocation ATPase. A number of mutants showed strong correlations between in vivo protein export ability, in vitro translocation activity and in vitro translocation ATPase activity, substantiating the importance of SecY irt vivo and in vitro. A few other mutants were affected in only one or two aspects of these properties, suggesting that they were differentially affected in some substeps of translocation. These results provide further evidence that SecY has vital roles in protein translocation, in which the 'motor' function of SecA and the 'channel' function of SecYEG should be coordinated.

We used hexahistidine-tagged SecE and SecY to study how the core subunits (SecY, SecE and SecG) of Escherichia coli protein translocase interact with each other. Detergent extracts were prepared from the plasma membranes and fractionated by Ni2+-NTA agarose affinity binding. Although His(6)-SecE, expressed in wild-type cells, brought down both SecY and SecG, neither of them was brought down when the same protein was expressed in the secY24 mutant cells. His(6)-SecY brought down both SecE and SecG, as expected. Interestingly, His(6)-SecY24 was able to bring down SecG but not SecE. These results confirm our previous conclusion that the secY24 alteration impairs the SecY-SecE interaction, and demonstrate that SecY and SecG can form a complex that does not contain SecE. Likewise, SecY-SecE complex could be isolated from the secG-deleted strain. The trimeric complex, in detergent extracts, dissociated at a critical temperature between 23 and 26 degrees C, whereas the SecY-SecE complex without SecG dissociated at a slightly lower temperature (20-23 degrees C). We conclude that each of SecE and SecG independently binds to SecY, the central subunit of protein translocase, although the trimeric complex is more stable than the binary complexes. (C) 1997 Federation of European Biochemical Societies.

Alkaline phosphatase of Escherichia coli (a homodimeric protein found in the periplasmic space) contains two intramolecular disulfide bonds (Cys-168-Cys-178 and Cys-286-Cys-336) that are formed after export to the periplasmic space. The location-specific folding character of this enzyme allowed its wide usage as a reporter of protein localization in prokaryotic cells. To study the roles of disulfide bonds in alkaline phosphatase, we eliminated each of them by Cys to Ser mutations. Intracellular stability of alkaline phosphatase decreased in the absence of either one or both of the disulfide bonds. The mutant proteins were stabilized in a DegP protease-deficient strain, allowing accumulation at significant levels and subsequent characterization. A mutant protein that lacked the N-terminally located disulfide bond (Cys-168-Cys-178) was found to have Cys-286 and Cys-336 residues disulfide-bonded, to have a dimeric structure, and to have almost full enzymatic activity. Nevertheless, the mutant protein lost the trypsin-resistant conformation that is characteristically observed for the wild-type enzyme. In contrast, mutants lacking Cys-286 and Cys-336 were monomeric and inactive. These results indicate that the Cys-286-Cys-336 disulfide bond is required and is sufficient for correctly positioning the active site region of this enzyme, but such an active conformation is still insufficient for the conformational stability of the enzyme. Thus, a fully active state of this enzyme can be formed without full protein stability, and the two disulfide bonds differentially contribute to these properties.

We examined in vitro translocation of pro-OmpA derivatives with a His(6) tag at various positions in their mature proteins and with a c-Myc tag at their C termini across inverted membrane vesicles of Escherchia coil, Those with a His(6) tag in the N-terminal region of the mature domain, which corresponds to the ''translocation initiation domain'' proposed previously (Andersson, H., and von Heijne, G. (1991) Proc. Natl. Acad. Sci. U.S.A. 88, 9751-9754), could not be translocated in the presence of 100 mu M Ni2+, while OmpA derivatives with a His, tag in the middle of or at the C terminus did not show such Ni2+ sensitivity. The inhibitory action of Ni2+ on pro-3His-OmpA' (with a His(6) tag after the third amino acid of the mature OmpA-c-Myc region) translocation was exerted only during early events, after which it became ineffective, The inhibition point of Ni2+ was suggested to lie between membrane targeting and exposure of the signal cleavage site to the periplasm since the unprocessed and membrane-bound form of pro-3His-OmpA' was accumulated by the addition of Ni2+, The Ni2+-''trapped'' precursor was released from its translocation block by 30 mM histidine, which should compete with the His, tag on the precursor protein for formation of a Ni2+ chelating complex, We propose that Ni2+ confers a reversible positive charge effect on the His(6)-tagged initiation domain of the pro-OmpA derivatives and inhibits an early event(s) of protein translocation, such as presentation of the precursor to the membranous part of the translocase, This system will be useful in dissecting early events of the protein translocation pathway.

Intracellular stability of alpha fragments of beta-galactosidase in Escherichia coli has been studied by pulse-chase/immunoprecipitation experiments. An alpha fragment encoded by the pUC118 vector was relatively stable with an estimated half-life of about 12 min at 37 degrees C, whereas another vector, pSTV28, encoded a less stable alpha fragment that had a different carboxy-terminal sequence. Stability of the fragment was found to be affected markedly by amino-terminal attachment of other sequences. An amino-terminal fusion of a sequence derived from cytoplasmic domain 4 of the SecY protein shortened the half-life of the alpha fragment to less than 1 min. In contrast, an amino-terminal sequence from the NusG protein had no apparent effect on the stability of the fragment. In a fusion protein in which the intact SecY protein was fused to the alpha fragment, stabilization of the SecY part by overproduction of the partner SecE protein resulted in an increased alpha complementation activity of beta-galactosidase. These results indicate that stability of alpha fragment can be dictated by the stability of the fused protein. The alpha fragment of beta-galaclosidase, which is unique in that it is largely unstructured but can be ''active'' in alpha complementation, may be used as an in vivo indicator of stability of proteins attached to it. (C) 1995 Academic Press, Inc.

The FtsH (HflB) protein of Escherichia coli is inte grated into the membrane with two N-terminally located transmembrane segments, while its large cytoplasmic domain is homologous to the AAA family of ATPases, The previous studies on dominant negative ftsH mutants raised a possibility that FtsH functions in multimeric states, We found that FtsH was eluted at fractions corresponding to a larger molecular weight than expected from monomeric structure in size-exclusion chromatography, Moreover, treatment of membranes or their detergent extracts with a cross-linker, dithiobis(succinimidyl propionate), yielded cross linked products of FtsH, To dissect possible FtsH complex, we constructed an FtsH derivative with c-Myc epitope at its C terminus (FtsH-His(6)-Myc). When membranes prepared from cells in which FtsH-His(6)-Myc was overproduced together with the normal FtsH were treated with the cross-linker, intact FtsH and in vitro degradation products of FtsH-His(6)-Myc without the tag were cross linked with the tagged FtsH protein, Co-immunoprecipitation experiments confirmed the interaction between the FtsH molecules, To identify regions of FtsH required or sufficient for this interaction, we constructed chimeric proteins between FtsH and EnvZ, a protein with a similar topological arrangement, by exchanging their corresponding domains, We found that only the FtsH-EnvZ hybrid protein with an FtsH-derived membrane anchoring domain and an EnvZ-derived cytoplasmic domain caused a dominant ftsH phenotype and was cross-linked with FtsH, We suggest that the N-terminal transmembrane region of FtsH mediates directly the interaction between the FtsH subunits.

A mutant form of SecY, SecY(-d)1, was previously suggested to sequester a component(s) of the protein translocator complex. Its synthesis from a plasmid leads to interference with protein export in Escherichia coli. SecE is a target of this sequestration, and its overproduction cancels the export interference. We now report that overexpression of another gene, termed syd, also suppresses secY(-d)1. The nucleotide sequence of syd predicted that it encodes a protein of 181 amino acid residues, which has been identified by overproduction, purification, and determination of the amino terminal sequence. Cell fractionation experiments suggested that Syd is loosely associated with the cytoplasmic surface of the cytoplasmic membrane. SecY may be involved in the membrane association of Syd since the association is saturable, the extent of which depends on the overproduction of SecY. SecY is rapidly degraded in vivo unless its primary partner, SecE, is sufficiently available. Overproduction of Syd was found to stabilize oversynthesized SecY. However, Syd cannot stabilize the SecY(-d)1 form of SecY. Thus, in the presence of both secY(+) and secY(-d)1, Syd increases the effective SecY(+)/SecY(-d)1 ratio in the cell and cancels the dominant interference by the latter. We also found that overproduction of Syd dramatically inhibits protein export in the secY24 mutant cell in which SecY-SecE interaction has been weakened. These results indicate that Syd, especially when it is overproduced, has abilities to interact with SecY. Possible significance of such interactions is discussed in conjunction with the apparent lack of phenotypic consequences of genetic disruption of syd.

The binding and hydrolysis of guanosine triphosphate (GTP) by the small GTP-binding protein Sar1p is required to form transport vesicles from the endoplasmic reticulum (ER) in Saccharomyces cerevisiae. Experiments revealed that an interaction between Sar1p and the Sec23p subunit of an oligomeric protein is also required for vesicle budding. The isolated Sec23p subunit and the oligomeric complex stimulated guanosine triphosphatase (GTPase) activity of Sar1p 10- to 15-fold but did not activate two other small GTP-binding proteins involved in vesicle traffic (Ypt1p and ARF). Activation of GTPase was inhibited by an antibody to Sec23p but not by an antibody that inhibits the budding activity of the other subunit of the Sec23p complex. Also, activation was thermolabile in pure samples of Sec23p that were isolated from two independent sec23 mutant strains. It appears that Sec23p represents a new class of GTPase-activating protein because its sequence shows no similarity to any known member of this family.

A cell-free protein transport reaction has been used to monitor the purification of a functional form of the Sec23 protein, a SEC gene product required for the formation or stability of protein transport vesicles that bud from the endoplasmic reticulum (ER). Previously, we reported that Sec23p is an 84-kDa peripheral membrane protein that is released from a sedimentable fraction by vigorous mechanical agitation of yeast cells and is required for ER to Golgi transport assayed in vitro. We have purified soluble Sec23p by complementation of an in vitro ER to Golgi transport reaction reconstituted with components from sec23 mutant cells. Sec23p overproduced in yeast exists in two forms: a monomeric species and a species that behaves as a 250- to 300-kDa complex that contains Sec23p and a distinct 105-kDa polypeptide (p105). Sec23p purified from cells containing one SEC23 gene exists solely in the large multimeric form. A stable association between Sec23p and p105 is confirmed by cofractionation of the two proteins throughout the purification. p105 is a novel yeast protein involved in ER to Golgi transport. Like Sec23p, it is required for vesicle budding from the ER because p105 antiserum completely inhibits transport vesicle formation in vitro.