Tohru Yoshihisa

Roles of Disulfide Bonds in Bacterial Alkaline Phosphatase

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.

SecY and SecA interact to allow SecA insertion and protein translocation across the Escherichia coli plasma membrane

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.

FtsH, a Membrane-bound ATPase, Forms a Complex in the Cytoplasmic Membrane of Escherichia coli

The FtsH (HflB) protein of Escherichia coli is integrated 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-His6-Myc). When membranes prepared from cells in which FtsH-His6-Myc was overproduced together with the normal FtsH were treated with the cross-linker, intact FtsH and in vitro degradation products of FtsH-His6-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.

Cytoplasmic splicing of tRNA in Saccharomyces cerevisiae

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.

Roles of TOM70 in import of presequence-containing mitochondrial proteins

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.

Handling tRNA introns, archaeal way and eukaryotic way

Introns 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.

A novel import route for an N‐anchor mitochondrial outer membrane protein aided by the TIM23 complex

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 (ΔΨ). Therefore, Om45 is anchored to the OM via the IMS by a novel import pathway involving the TIM23 complex.

Subunit interactions in the Escherichia coli protein translocase: SecE and SecG associate independently with SecY

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 His6‐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. His6‐SecY brought down both SecE and SecG, as expected. Interestingly, His6‐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°C, whereas the SecY‐SecE complex without SecG dissociated at a slightly lower temperature (20–23°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.

Dual Functions of Yeast tRNA Ligase in the Unfolded Protein Response: Unconventional Cytoplasmic Splicing of HAC1 Pre-mRNA Is Not Sufficient to Release Translational Attenuation

Unconventional cytoplasmic splicing of HAC1 mRNA is essential for the yeast unfolded protein response (UPR). The UPR requires translational regulation of unspliced and spliced forms of HAC1 mRNAs. Here we report that tRNA ligase, Rlg1p, which ligates HAC1 exons in its splicing, has another face as a translational regulator of HAC1 mRNA.

Sec-dependent Pathway and ΔpH-dependent Pathway Do Not Share a Common Translocation Pore in Thylakoidal Protein Transport

Thylakoidal proteins of plant chloroplasts are transported to thylakoids via several different pathways, including the ΔpH-dependent and the Sec-dependent pathways. In this study, we asked if these two pathways utilize a common translocation pore. A fusion protein consisting of a 23-kDa subunit of the oxygen evolving complex and Escherichia coli biotin carboxyl carrier protein was biotinylated in E. coli cells and purified. When incubated with isolated pea thylakoids in the absence of avidin, the purified fusion protein was imported into the thylakoids via the ΔpH-dependent pathway. However in the presence of avidin, the fusion protein became lodged in the thylakoid membranes, with its N terminus reaching the thylakoidal lumen, while its C-terminal segment complexed with avidin exposed on the thylakoidal surface. The translocation intermediate of the fusion protein inhibited the import of authentic 23-kDa subunit, suggesting that it occupies a putative translocation pore for the ΔpH-dependent pathway. However the intermediate did not block import of the 33-kDa subunit of the oxygen evolving complex, which is a substrate for the Sec-dependent pathway. These results provide evidence against the possibility of a common translocation pore shared by the Sec-dependent pathway and the ΔpH-dependent pathway.