摘要:

SummaryEukaryotic cells have evolved a complex set of intracellular organelles, each of which possesses a specific complement of enzymes and performs unique metabolic functions. This compartmentalization of cellular functions provides a level of metabolic control not available to prokaryotes. However, it presents the eukaryotic cell with the problem of targeting proteins to their specific location (s). Proteins must be efficiently transported from their site of synthesis in the cytosol to their specific organelle (s). Such a process may require translocation across one or more hydrophobic membrane barriers and/or asymmetric integration into specific membranes.Proteins carrycis‐acting amino acid sequences that serve to act as recognition motifs for protein sorting and for the cellular translocation machinery. Sequences that target proteins to the endoplasmic reticulum/ secretory pathway, mitochondria, and chloroplasts are often present as cleavable amino‐terminal extensions. In contrast, most peroxisomal proteins are synthesized at their mature size and are translocated to the organelle without any post‐translational modification. This review will summarize what is known about how yeast solve the problem of specifically importing proteins into peroxisomes and will suggest future directions for investigations into peroxisome biogenesis in

ISSN:0950-382X    

DOI:10.1111/j.1365-2958.1992.tb01780.x

出版商:Blackwell Publishing Ltd    

年代:1992

数据来源: WILEY

摘要:

SummarySelenocysteine synthase ofEscherichia colicatalyses the biosynthesis of selenocysteine in the form of the aminoacyl‐RNA complex, the reaction intermediate being aminoacrylyl‐tRNAseccovalently bound to the prosthetic group of the enzyme. Selenocysteine synthase and the specific aminoacrylyl‐tRNAsec‐enzyme complex as well as the isolated seryl‐tRNAsecwere investigated in the electron microscope and analysed by means of image processing to a resolution of 2 nm in projection. The stoichiometric composition of the selenocysteine synthase molecule was elucidated by scanning transmission electron microscopic mass determination. The enzyme has a fivefold symmetric structure and consists of 10 monomers arranged in two rings. The tRNA is bound near the margin of the dimeric subunits. Principal component analysis of the tRNA‐enzyme complexes revealed that the selenocysteine synthase appears to bind only one seryl‐tRNAsecper dimer, which is consistent with the result of biochemical bi

ISSN:0950-382X    

DOI:10.1111/j.1365-2958.1992.tb01781.x

出版商:Blackwell Publishing Ltd    

年代:1992

数据来源: WILEY

摘要:

SummaryTheSAL4gene of the yeastSaccharomyces cerevisiaeencodes a novel translation factor (Sal4p) involved in maintaining translational fidelity. Using a polyclonal antibody raised against a Sal4p‐β‐galac‐tosidase fusion protein, Sal4p was shown to be almost exclusively associated with the ribosomal fraction. Even when the ribosomes were treated with 0.8 M KCl, only low levels of Sal4p were detected in the post‐ribosomal supernatant, suggesting a very strong affinity between Sal4p and the ribosome. Analysis of the distribution of Sal4p in the ribosomal population revealed that it was principally associated with 40S subunits, monosomes and polysomes. Incubation in high salt concentrations (0.8 M KCl) suggested that the affinity of Sal4p for the 40S subunit was lower than that for monosomes or polysomes. The Sal4p:ribosome association was only maintained when ribosomes were prepared in the presence of the translation elongation inhibitor cycloheximide; in uninhibited cells much lower levels of Sal4p were detectable in the ‘run‐off’ polysomes. In view of these data, and given the stoichiometry of Sal4p to individual ribosomal proteins (estimated at less than 1:20), we suggest that Sal4p plays an ancillary role in translati

ISSN:0950-382X    

DOI:10.1111/j.1365-2958.1992.tb01782.x

出版商:Blackwell Publishing Ltd    

年代:1992

数据来源: WILEY

摘要:

SummaryTheRhizobium melilotgenesdctBanddctDpositively regulate the expression ofdctA, which encodes a C4‐dicarboxylate transport protein. Here we characterize an element (UAS) located upstream ofdctAthat has tandem binding sites for thedctDgene product (DctD). At relatively low concentrations of active DctD, the element activateddctAtranscription, but at relatively high concentrations of DctD it was inhibitory. The UAS failed to function when placed further upstream ofdctA.Both DctD‐binding sites were required for optimal UAS function, despite a 50‐ to 100‐fold difference in binding affinities. Moving the promoter distal binding site 5 bp further upstream was functionally equivalent to its deletion. Based on these data, we hypothesize that the α54‐dependent activator DctD binds co‐operatively to the R.meliloti dctAUAS, and that occupancy of both sites is required for maximal activ

ISSN:0950-382X    

DOI:10.1111/j.1365-2958.1992.tb01783.x

出版商:Blackwell Publishing Ltd    

年代:1992

数据来源: WILEY

摘要:

SummaryStrains ofNeisseria meningitidisexpress one of two porin proteins. These porins have been identified as the class 2 and class 3 proteins, and express serotype‐specific epitopes. The gene for the class 3 protein was amplified by the polymerase chain reaction from the DNA of a serotype 4 strain as a 1025bp fragment. The nucleotide sequence of this gene was determined and compared with two recently published sequences. On the basis of this comparison we have identified two major variable regions in the translated protein sequence, VR1 and VR2, that may be associated with serotype specificity. Three other variable regions were also identified. The sequences in the VR1 and VR2 regions from five additional group BN. meningitidisstrains of serotypes 1, 4, 8,12, and 15, all expressing class 3 proteins, were determined. The VR1 and VR2 regions were variable and were flanked by highly conserved regions among eight different class 3 sequences. These two variable regions of 15 and 9 amino acids are predicted to be in surface‐exposed lo

ISSN:0950-382X    

DOI:10.1111/j.1365-2958.1992.tb01784.x

出版商:Blackwell Publishing Ltd    

年代:1992

数据来源: WILEY

摘要:

SummaryBy using deletional analysis the origin of replication,oriR, of the streptococcal plasmid plP501 inBacillus subtilishas been mapped at a position immediately downstream of therepRgene. Determination of both the right and left border oforiRallowed the definition of a sequence of a maximum of 52 nucleotides which theoretically constitutes the minimal origin of replication. Recently, the start point of leading‐strand synthesis of the closely related plasmid pAMβ1 has been mapped at a position which is located exactly in the middle of this sequence (Bruandet al., 1991). The function ofohRdid not depend on its location downstream of therepRgene. Translocation oforiRcontaining fragments to other regions of the plasmid proved to be possible. The smallest translocated fragment that still reconstituted autonomous replication was 72bp in size. This fragment was also active in directing the replication of anEscherichia coliplasmid inB. subtiliswhen the RepR protein was suppliedin transfrom arepRgene integrated into the host chromosome. The transformation efficiency of plasmids carrying translocatedoriRfragments showed a certain dependence on the fragment length and orientation. The DNA sequence oforiRincluded an inverted repeat, both branches of which appeared to be essential fororiRfunction. The repeats oforiRshared sequence similarity with a repeat located upstream of promoter pll, which has been suggested to be involved in autoregulation ofrepRexpressi

ISSN:0950-382X    

DOI:10.1111/j.1365-2958.1992.tb01785.x

出版商:Blackwell Publishing Ltd    

年代:1992

数据来源: WILEY

摘要:

SummaryThe potassium‐translocating Kdp‐ATPase ofEscherichia colishares common functional properties with eukaryotic P‐type ATPases. The KdpB subunit has been identified as the catalytic subunit forming the phosphorylated intermediate. Substitution of Asp 307 in KdpB by Glu, Asn, Gin, Tyr, His, Ala or Ser by site‐directed mutagenesis and the subsequent transfer of the point mutations to the chromosome revealed that the mutants were not functioning with respect to cell growth at low K’ concentrations and ATPase activity as well as phosphorylation capacity of the purified Kdp complex. These findings indicate that Asp‐307 in KdpB is the phosphorylation site of the Kdp‐ATPase. In contrast, replacement of the close but non‐conserved Asp‐300 by Asn or Glu has no immediate influence on the enzyme functions tested. However, the Km for K+of the ATPase activity has been increased 30‐fold compared with t

ISSN:0950-382X    

DOI:10.1111/j.1365-2958.1992.tb01786.x

出版商:Blackwell Publishing Ltd    

年代:1992

数据来源: WILEY

摘要:

SummaryThe methanol‐soluble, heat‐stable enterotoxin ofEscherichia coliis a protease‐resistant extracellular peptide which is synthesized as a 72‐amino‐acid precursor Pre‐Pro‐STA3. The specific roles of Fre (19 amino acids), Pro (34 amino acids) and STA3(19 amino acids) in the secretion process were studied by functionally deleting each of the three domains. Deletion of the Pre signal sequence resulted in a short‐lived cell‐associated molecule with an Mrequivalent to that of Pro‐STA3.Deletion of Pro (i.e., Pre‐STA3) resulted in the rapid extracellular accumulation of STA3the periplasmic intermediate found in the secretion of the wild‐type toxin was undetected. Deletion of the STA3domain resulted in a cell‐associated Pre‐Pro peptide; with time this form converted to periplasmic Pro which later became extracellular. When DNA encoding either STA3, by itself, or Pro‐STA3 (lacking the signal peptide) was expressed, these peptides were degraded intracellularly, with no periplasmic or extracellular forms detected.The results presented demonstrate that the signal peptide (Pre) is essential even for the export of small peptides to the periplasm, and that its absence causes the STAS domain to become susceptible to intracellular proteases. The rapid degradation of intracetlular STA3indicates that its proteolytic resistance is acquired in a compartment other than the cytoplasm. The results also show that after the Pre domain is proteolytically cleaved from Pre‐STA3and Pre‐Pro, the STA3 and Pro peptides can exit to the culture supernatant. Since STA3and Pro have neither sequence nor c

ISSN:0950-382X    

DOI:10.1111/j.1365-2958.1992.tb01787.x

出版商:Blackwell Publishing Ltd    

年代:1992

数据来源: WILEY

摘要:

SummaryIt has been shown that retrons, retro‐elements in bacteria, produce a reverse transcriptase (RT) and multicopy single‐stranded DNA(msDNA) whose 5′ end is covalently linked to RNA (msdRNA) by a 2′‐5′ phosphodiester bond. Here, I show that a retron in clinicalEscherichia colistrain 161 produces an msDNA unlinked to RNA. The msDNA produced by this retron is a 79‐nucleotlde‐long single‐stranded DNA with monophosphate on its 5′ terminus. When the retron in strain 161 is cloned intoE. coliK‐12, the majority of msDNA produced in the clone is the same as the msDNA in the clinical strain. However, in the K‐12 clone, about 10% of the msDNA produced is present as a DNA covalently linked to RNA. The DNA part of this RNA‐DNA compound is an 83 nucleotides long with the same sequence as the unbranched msDNA, except for the presence of four additional nucleotides at the 5′ side. From the analysis of the RNA‐DNA compound and the results ofin vitrosynthesis, I show that the primary product of reverse transcription in this retron is an 83‐nucleotide‐tong DNA covalently linked to RNA. This RNA‐DNA compound is further processed to the final product, the 79‐nucleotide‐long msDNA with a terminal 5′ monophosphate, by an endonucleolytic cleavage between the fourth and fifth positions of the DNA component of the RNA‐DNA compound. The minimum region required for the production of such msDNA free of RNA contains only genes known to be required for the synthesis of branched msDNA‐RNA compound in other retrons(msd, msrandret). This suggests that either the RT has an endonuclease activity or that the msDNA

ISSN:0950-382X    

DOI:10.1111/j.1365-2958.1992.tb01788.x

出版商:Blackwell Publishing Ltd    

年代:1992

数据来源: WILEY

摘要:

SummaryA series of deletions introduced into thegvpgene cluster ofHaloferax mediterranel, comprising 14 genes involved in gas vesicle synthesis (mc‐vac‐region), was investigated by transformation experiments. Gas vesicle production and the expression of thegvpAgene encoding the major gas vesicle protein, GvpA, was monitored in eachHaloferax volcaniitransformant. Whereas transformants containing the entire mc‐vac‐region produced gas vesicies (Vac+), various deletions in the region 5′ togvpA(encompassinggvpD‐gvpM) or 3′ togvpA(containinggvpC, gvpNandgvpO) revealed Vac−transformants. All these transformants expressedgvpAand contained the 8 kDa GvpA protein as shown by Western analysis. However, transformants containing thegvpAgene by itself indicated a lower level of GvpA than observed with each of the other transformants. None of these transformants containing deletion constructs assembled the GvpA protein into gas vesicles. In contrast, transformants containing a construct carrying a 918 bp deletion internal togvpDexhibited a tremendous gas vesicle overproduction, suggesting a regulatory role for thegvpDgene or Its product. This is the first assignment of a functional role for one of the 13 halobacterialgvpgenes found in addition togvpAthat are involved in the synthesis of this u

ISSN:0950-382X    

DOI:10.1111/j.1365-2958.1992.tb01789.x

出版商:Blackwell Publishing Ltd    

年代:1992

数据来源: WILEY