Reconstitution of protein translocation from solubilized yeast membranes reveals topologically distinct roles for BiP and cytosolic Hsc70

We reconstituted prepro-alpha-factor translocation and signal peptide processing using a yeast microsomal detergent soluble fraction formed into vesicles with soybean phospholipids. Reconstituted translocation required ATP, and was deficient when sec63 and kar2 (BiP) mutant cells were used as a source of membranes. Normal translocation was observed with vesicles reconstituted from a mixture of pure wild-type yeast BiP and a soluble fraction of kar2 mutant membranes. Two other heat-shock cognate (hsc) 70 homologs, yeast cytosolic hsc70 (Ssalp) and E. coli dnaK protein did not replace BiP. Conversely, BiP was not active under conditions where translocation into native ER vesicles required cytosolic hsc70. We conclude that cytosolic hsc70 and BiP serve noninterchangeable roles in polypeptide translocation, possibly because distinct, asymmetrically oriented membrane proteins are required to recruit each protein to opposing surfaces of the ER membrane.     

Structure and evolution of a group of related aminoacyl-tRNA synthetases

A yeast nuclear gene, designated MSK1, has been selected from a yeast genomic library by transformation of a respiratory deficient mutant impaired in acylation of mitochondrial lysine tRNA. This gene confers a respiratory competent phenotype and restores the mutant's ability to acylate the mitochondrial lysine tRNA. The amino acid sequence of the protein encoded by MSK1 is homologous to yeast cytoplasmic lysyl-tRNA synthetase and to the product of the herC gene, which has recently been suggested to code for the Escherichia coli enzyme. These observations indicate that MSK1 codes for the lysyl-tRNA synthetase of yeast mitochondria. Several regions of high primary sequence conservation have been identified in the bacterial and yeast lysyl-tRNA synthetases. These domains are also present in the aspartyl- and asparaginyl-tRNA synthetases, further confirming the notion that all three present-day enzymes originated from a common ancestral gene. The most conserved domain, located near the carboxyl terminal ends of this group of synthetases is characterized by a cluster of glycines and is also highly homologous to the carboxyl-terminal region of the E. coli ammonia-dependent asparagine synthetase. A catalytic function of the carboxyl terminal domain is indicated by in vitro mutagenesis of the yeast mitochondrial lysyl-tRNA synthetase. Replacement of any one of three glycine residues by alanine and in one case by aspartic acid completely suppresses the activity of the enzymes, as evidenced by the inability of the mutant genes to complement an msk1 mutant, even when present in high copy. Other mutations result in partial loss of activity. Only one glycine replacement affects the stability of the protein in vivo. The observed presence of a homologous domain in asparagine synthetase, which, like the aminoacyl-tRNA synthetases, catalyzes the formation of an aminoacyladenylate, suggests that the glycine-rich sequence is part of a catalytic site involved in binding of ATP and of the aminoacyladenylate intermediate.     

Role of autolysins in the EDTA-induced lysis of Pseudomonas aeruginosa

Abstract A DNA fragment containing the genes secE, nusG and rplK of Staphylococcus carnosus was cloned using the Escherichia coli rplK gene as a probe. The S. carnosus secE homologue encodes a protein of 65 amino acid residues which is homologous to the carboxyl-terminal region of the E. coli SecE protein. The S. carnosus SecE polypeptide which, in contrast to the E. coli SecE protein, contains only one putative transmembrane segment, could fully replace the E. coli SecE protein in two different secE mutants. These results strongly suggest that the identified secE gene encodes an important component of the S. carnosus protein export apparatus.     

Mutations in the signal sequence of prepro-alpha-factor inhibit both translocation into the endoplasmic reticulum and processing by signal peptidase in yeast cells.

The effects of five single-amino-acid substitution mutations within the signal sequence of yeast prepro-alpha-factor were tested in yeast cells. After short pulse-labelings, virtually all of the alpha-factor precursor proteins from a wild-type gene were glycosylated and processed by signal peptidase. In contrast, the signal sequence mutations resulted in the accumulation of mostly unglycosylated prepro-alpha-factor after a short labeling interval, indicating a defect in translocation of the protein into the endoplasmic reticulum. Confirming this interpretation, unglycosylated mutant prepro-alpha-factor in cell extracts was sensitive to proteinase K and therefore in a cytosolic location. The signal sequence mutations reduced the rate of translocation into the endoplasmic reticulum by as much as 25-fold or more. In at least one case, mutant prepro-alpha-factor molecules were translocated almost entirely posttranslationally. Four of the five mutations also reduced the rate of proteolytic processing by signal peptidase in vivo, even though the signal peptide alterations are not located near the cleavage site. This study demonstrates that a single-amino-acid substitution mutation within a eucaryotic signal peptide can affect both translocation and proteolytic processing in vivo and may indicate that the recognition sequences for translocation and processing overlap within the signal peptide.     

Yeast gene which suppresses the defect in protein export of a secY mutant of E. coli

To find factors participating in protein translocation in yeast, we screened a yeast genomic library for genes which, when introduced into Escherichia coli, suppressed secY24, a temperature sensitive mutation of an essential integral membrane protein (SecY) required for protein export. We isolated and characterized a gene (YSY6) which improved the translocation of the OmpA protein in mutant strain IQ85(secY24). It could also suppress another mutant [rplO215(Am)], in which the level of expression of the SecY protein is decreased at high-temperature. The YSY6 gene encodes a small amphiphilic peptide consisting of 65 amino acids, which can be expressed in E. coli cells.     

Defective Escherichia coli signal peptides function in yeast

To investigate structural characteristics important for eukaryotic signal peptide function in vivo, a hybrid gene with interchangeable signal peptides was cloned into yeast. The hybrid gene encoded nine residues from the amino terminus of the major Escherichia coli lipoprotein, attached to the amino terminus of the entire mature E. coli beta-lactamase sequence. To this sequence were attached sequences encoding the nonmutant E. coli lipoprotein signal peptide, or lipoprotein signal peptide mutants lacking an amino-terminal cationic charge, with shortened hydrophobic core, with altered potential helicity, or with an altered signal-peptide cleavage site. These signal-peptide mutants exhibited altered processing and secretion in E. coli. Using the GAL10 promoter, production of all hybrid proteins was induced to constitute 4-5% of the total yeast protein. Hybrid proteins with mutant signal peptides that show altered processing and secretion in E. coli, were processed and translocated to a similar degree as the non-mutant hybrid protein in yeast (approximately 36% of the total hybrid protein). Both non-mutant and mutant signal peptides appeared to be removed at the same unique site between cysteine 21 and serine 22, one residue from the E. coli signal peptidase II processing site. The mature lipo-beta-lactamase was translocated across the cytoplasmic membrane into the yeast periplasm. Thus the protein secretion apparatus in yeast recognizes the lipoprotein signal sequence in vivo but displays a specificity towards altered signal sequences which differs from that of E. coli.     

Lactose transport system of Streptococcus thermophilus. The role of histidine residues.

The lactose transport protein (LacS) of Streptococcus thermophilus is a chimeric protein consisting of an amino-terminal carrier domain and a carboxyl-terminal phosphoenolpyruvate:sugar phosphotransferase system (PTS) IIA protein domain. The histidine residues of LacS were changed individually into glutamine or arginine residues. Of the 11 histidine residues present in LacS, only the His-376 substitution in the carrier domain significantly affected sugar transport. The region around His-376 was found to exhibit sequence similarity to the region around His-322 of the lactose transport protein (LacY) of Escherichia coli, which has been implicated in sugar binding and in coupling of sugar and H+ transport. The H376Q mutation resulted in a reduced rate of uptake and altered affinity for lactose (beta-galactoside), melibiose (alpha-galactoside), and the lactose analog methyl-beta-D-thiogalactopyranoside. Similarly, the extent of accumulation of the galactosides by cells expressing LacS(H376Q) was highly reduced in comparison to cells bearing the wild-type protein. Nonequilibrium exchange of lactose and methyl-beta-D-thiogalactopyranoside by the H376Q mutant was approximately 2-fold reduced in comparison to the activity of the wild-type transport protein. The data indicate that His-376 is involved in sugar recognition and is important, but not essential, for the cotransport of protons and galactosides. The carboxyl-terminal domain of LacS contains 2 histidine residues (His-537 and His-552) that are conserved in seven homologous IIA protein(s) (domains) of PTSs. P-enolpyruvate-dependent phosphorylation of wild-type LacS, but not of the mutant H552Q, was demonstrated using purified Enzyme I and HPr, the general energy coupling proteins of the PTS, and inside-out membrane vesicles isolated from E. coli in which the lactose transport gene was expressed. The His-537 and His-552 mutations did not affect transport activity when the corresponding genes were expressed in E. coli.     

Comparison of the lipoprotein gene among the enterobacteriaceae. DNA sequence of Morganella morganii lipoprotein gene and its expression in Escherichia coli

A DNA sequence of 532 base pairs encompassing the entire Morganella morganii lipoprotein gene (lpp) was determined. Sequence comparisons of the M. morganii lpp gene with the lpp genes from Escherichia coli, Serratia marcescens, and Erwinia amylovora reveal that the M. morganii lpp gene is more distantly related to the E. coli lpp gene than any of the other lpp genes examined. Between the E. coli and M. morganii lpp genes, the following homologies were found: 44% in the promoter region (bases, -45 to -1), 88% in the 5'-end untranslated region of the mRNA, 58% in the signal sequence coding region, 75% in the coding region for the first 51 and 43% for the last 7 amino acid residues. Upstream of the promoter region and downstream of the termination codon, there are extensive insertions, deletions, and base substitutions. In spite of the differences in the DNA sequences, the lipoprotein structure was found to be highly conserved except for the carboxyl-terminal sequence of 7 amino residues. The coding region of the M. morganii lpp gene including the signal sequence was inserted into an expression cloning vector so that the production of the M. morganii lipoprotein could be induced in E. coli by a lac inducer, isopropyl-beta-D-thioglactoside. It was found that when induced, the M. morganii prolipoprotein was apparently secreted normally across the E. coli cytoplasmic membrane, modified with glycerol and palmitic acid, processed to the mature lipoprotein, and assembled in the E. coli outer membrane. The bound form covalently linked to the peptidoglycan was also found.     

The bacterial magnesium transporter CorA can functionally substitute for its putative homologue Mrs2p in the yeast inner mitochondrial membrane.

The yeast nuclear gene MRS2 encodes a protein of 54 kDa, the presence of which has been shown to be essential for the splicing of group II intron RNA in mitochondria and, independently, for the maintenance of a functional respiratory system. Here we show that the MRS2 gene product (Mrs2p) is an integral protein of the inner mitochondrial membrane. It appears to be inserted into this membrane by virtue of two neighboring membrane spanning domains in its carboxyl-terminal half. A large amino-terminal and a shorter carboxyl-terminal part are likely to be exposed to the matrix space. Structural features and a short sequence motif indicate that Mrs2p may be related to the bacterial CorA Mg2+ transporter. In fact, overexpression of the CorA gene in yeast partially suppresses the pet- phenotype of an mrs2 disrupted yeast strain. Disruption of the MRS2 gene leads to a significant decrease in total magnesium content of mitochondria which is compensated for by the overexpression of the CorA gene. Mutants lacking or overproducing Mrs2p exhibit phenotypes consistent with the involvement of Mrs2p in mitochondrial Mg2+ homeostasis.     

Vaccinia virus encodes an active thymidylate kinase that complements a cdc8 mutant of Saccharomyces cerevisiae.

A vaccinia virus open reading frame (ORF) previously predicted to encode thymidylate kinase (TmpK) is shown to encode an active enzyme. A copy of the ORF, generated by polymerase chain reaction, was cloned into an Escherichia coli inducible expression vector. Cell extracts of E. coli expressing the vaccinia gene contained high levels of TmpK activity, whereas extracts of cells without the TmpK gene did not. The vaccinia ORF expressed from a yeast vector complemented a Saccharomyces cerevisiae cdc8 mutant, demonstrating functional compatibility of the vaccinia virus and yeast TmpK enzymes. The gene is shown to be nonessential for the replication of vaccinia virus in cultured cells by the construction of a viable virus mutant that has the coding region of the TmpK gene interrupted by the Ecogpt gene. Synthesis of the vaccinia TmpK protein in infected cells was demonstrated by the use of a polyvalent rabbit antiserum raised against the purified TmpK enzyme expressed in E. coli to immunoprecipitate a 23-kDa early polypeptide from cells infected with wild type vaccinia but not from cells infected with the TmpK mutant. Plasmid vectors that allow the construction of recombinant viruses expressing foreign gene(s) from the nonessential TmpK locus are described.     

Secretion in yeast. Purification and in vitro translocation of chemical amounts of prepro-alpha-factor

The Saccharomyces cerevisiae mating pheromone precursor, prepro-alpha-factor, can be translocated across yeast endoplasmic reticulum membranes post-translationally in an in vitro system. This characteristic makes prepro-alpha-factor potentially useful as a probe in the biochemical dissection of the mechanism of this basic cellular process. Efforts have been limited by the inability to isolate sufficient quantities of such secretory protein precursors in a translocation-competent form. We report here the one-step purification of chemical amounts of translocation-competent prepro-alpha-factor using nickel ion affinity chromatography on nitrilotriacetate resin. An oligonucleotide encoding 6 histidine residues was inserted into a genomic clone encoding prepro-alpha-factor 5' of the naturally occurring translational stop codon by site-directed mutagenesis. The construct was expressed at high levels in a SecY- strain of Escherichia coli. The produced preprotein was solubilized in 6 M guanidine hydrochloride and bound to nitrilotriacetate resin. Prepro-alpha-factor was recovered at a purity in excess of 95% by elution with 0.25 M imidazole, 8 M urea, which competitively displaced the histidine affinity tag from the nickel column. The chemical amounts of prepro-alpha-factor obtained in this way were determined to be competent for translocation across yeast microsomal membranes and for subsequent modifications such as signal sequence cleavage and N-linked glycosylation.     

Expression in Escherichia coli of the Saccharomyces cerevisiae CCT gene encoding cholinephosphate cytidylyltransferase.

The coding region of the CCT gene from the yeast Saccharomyces cerevisiae was cloned into the pUC18 expression vector. The plasmid directed the synthesis of an active cholinephosphate cytidylyltransferase in Escherichia coli, confirming that CCT is the structural gene for this enzyme. The enzyme produced in E. coli efficiently utilized cholinephosphate and N,N-dimethylethanolaminephosphate, but N-methylethanolamine-phosphate and ethanolaminephosphate were poor substrates. Consistently, disruption of the CCT locus in the wild-type yeast cells resulted in a drastic decrease in activities with respect to the former two substrates. When activity was expressed in E. coli, over 90% was recovered in the cytosol, whereas most of the activity of yeast cells was associated with membranes, suggesting that yeast cells possess a mechanism that promotes membrane association of cytidylyltransferase.     

Secretion in yeast: structural features influencing the post-translational translocation of prepro-alpha-factor in vitro.

In vitro, efficient translocation and glycosylation of the precursor of yeast alpha-factor can take place post-translationally. This property of prepro-alpha-factor appears to be unique as it could not be extended to other yeast protein precursors such as preinvertase or preprocarboxypeptidase Y. In order to determine if specific domains of prepro-alpha-factor were involved in post-translational translocation, we carried out a series of experiments in which major domains were either deleted or fused onto reporter proteins. Fusion of various domains of prepro-alpha-factor onto the reporter protein alpha-globin did not allow post-translational translocation to occur in the yeast in vitro system. Prepro-alpha-factor retained its ability to be post-translationally translocated when parts or all of the pro region were deleted. Removal of the C-terminal repeats containing mature alpha-factor had the most profound influence as post-translational translocation decreased in proportion to the number of repeats deleted. Taken together, these results suggest that efficient post-translational translocation requires a signal sequence and the four C-terminal repeats. There does not however, appear to be specific information contained within the C-terminus, as their presence in fusion did not enable the post-translational translocation of reporter proteins. Lastly, the ability to post-translationally translocate radiochemically pure prepro-alpha-factor that had been isolated by immuno-affinity chromatography required the addition of a yeast lysate fraction. Moreover, post-translational translocation is a function of the microsomal membrane of yeast microsomes and not of a factor peculiar to the yeast lysate, as reticulocyte lysate supported this as well.     

The carboxyl-terminal tripeptide serine-lysine-leucine of firefly luciferase is necessary but not sufficient for peroxisomal import in yeast.

Firefly luciferase is imported into peroxisomes in insects, mammals, plants, and yeast, which implies that the mechanism of protein translocation into peroxisomes has been conserved during eukaryotic evolution. The carboxyl-terminal tripeptide serine-lysine-leucine in luciferase acts as a peroxisomal import signal in mammalian cells. We have investigated whether this tripeptide is also involved in translocation of firefly luciferase into peroxisomes in yeast (Saccharomyces cerevisiae). We show by gene fusion experiments that the carboxyl-terminal 104 amino acids of luciferase can direct a heterologous protein to yeast peroxisomes. Luciferase mutant proteins were tested for their ability to be imported into yeast peroxisomes in vivo. We demonstrate that mutations in the carboxyl-terminal serine-lysine-leucine tripeptide abolish translocation of the protein into yeast peroxisomes. However, when a passenger protein was tagged at its carboxyl terminus with this tripeptide the fusion protein did not go to peroxisomes. These results indicate that, in yeast, the tripeptide is necessary but not sufficient for peroxisomal import.     

A mutant Kex2 enzyme with a C-terminal HDEL sequence releases correctly folded human insulin-like growth factor-1 from a precursor accumulated in the yeast endoplasmic reticulum.

Mutations in the pro region of the yeast DNA hybrid of prepro-alpha-factor and human insulin-like growth factor-1 (IGF-1) cause the accumulation, in the yeast Saccharomyces cerevisiae, of an unglycosylated precursor protein where the pre sequence is missing. The prepro sequence of the prepro-alpha-factor consists of a pre or signal sequence and a proregion which possesses three sites for N-glycosylation. Isolation of a precursor, where the pro region is still linked to IGF-1 through a pair of dibasic amino acid residues, implies that the polypeptide may have translocated into the endoplasmic reticulum (ER) but has not been processed by the Golgi membrane-bound Kex2 endoprotease. However, the lack of any N-glycosylation in the translocated polypeptide is surprising. The mutated pro region, can be processed, in vitro, by treatment with a soluble form of the Kex2 enzyme. It is also possible to release the pro region, in vivo, by coexpressing a mutant Kex2 protease which is partially retained in the ER with the help of the C-terminal tetrapeptide sequence, HDEL. The mature IGF-1, which is secreted from the intracellular pool of precursor proteins, is predominantly an active, monomeric molecule, corroborating observations that early removal of the pro region before folding in the ER helps to prevent aberrant intermolecular disulfide-bond formation in IGF-1. These results have revealed the utility of the ER-retained Kex2 enzyme as a novel in vivo biochemical tool.     

The new gene mukB codes for a 177 kd protein with coiled-coil domains involved in chromosome partitioning of E. coli.

An Escherichia coli temperature sensitive mutant which produces spontaneously normal size anucleate cells at low temperature was isolated. The mutant is defective in a previously undescribed gene, named mukB, located at 21 min on the chromosome. The mukB gene codes for a large protein (approximately 180 kd). A 1534 amino acid protein (176,826 daltons) was deduced from the nucleotide sequence of the mukB gene. Computer analysis revealed that the predicted MukB protein has distinct domains: an amino-terminal globular domain containing a nucleotide binding sequence, a central region containing two alpha-helical coiled-coil domains and one globular domain, and a carboxyl-terminal globular domain which is rich in Cys, Arg and Lys. A 180 kd protein detected in wild-type cell extracts by electrophoresis is absent in mukB null mutants. Although the null mutants are not lethal at low temperature, the absence of MukB leads to aberrant chromosome partitioning. At high temperature the mukB null mutants cannot form colonies and many nucleoids are distributed irregularly along elongated cells. We conclude that the MukB protein is required for chromosome partitioning in E. coli.     

Molecular cloning, DNA structure and expression of the Escherichia coli D-xylose isomerase.

The D-xylose isomerase (EC 5.3.1.5) gene from Escherichia coli was cloned and isolated by complementation of an isomerase-deficient E. coli strain. The insert containing the gene was restriction mapped and further subcloning located the gene in a 1.6-kb Bg/II fragment. This fragment was sequenced by the chain termination method, and showed the gene to be 1002 bp in size. The Bg/II fragment was cloned into a yeast expression vector utilising the CYCl yeast promoter. This construct allowed expression in E. coli grown on xylose but not glucose suggesting that the yeast promoter is responding to the E. coli catabolite repression system. No expression was detected in yeast from this construct and this is discussed in terms of the upstream region in the E. coli insert with suggestions of how improved constructs may permit achievement of the goal of a xylose-fermenting yeast.     

The bimB3 mutation of Aspergillus nidulans uncouples DNA replication from the completion of mitosis.

A conditionally lethal mutation in the bimB gene of Aspergillus nidulans disrupts the normal regulatory patterns associated with mitotic events. This results in DNA replication in the absence of the completion of mitosis in the mutant at restrictive temperature. This defect yields large polyploid nuclei after several hours at restrictive temperature. The bimB gene has been cloned by genetic mapping and chromosome walking from the previously cloned amdS gene. The cloned DNA complements the temperature-sensitive recessive bimB3 mutation. Sequence analysis of overlapping complementary DNA clones for bimB predicts a polypeptide of 2,068 amino acids. The predicted polypeptide of 227,958 Da is shown to have a carboxyl-terminal region similar to those of the budding yeast ESP1 and fission yeast cut1+ genes. In contrast these genes exhibit no other regions of similarity to one another. The conserved domain in these three proteins and the similarity of the terminal mutant phenotypes for these genes are suggestive of a conserved function for this domain in each of the predicted polypeptides. We also present evidence for a second gene in the genome of A. nidulans which also has this conserved carboxyl-terminal region, suggesting that bimB, ESP1, and cut1+ may be members of a small gene family.