Escherichia coli SecB stimulates export without maintaining export competence of ribose-binding protein signal sequence mutants.

Ribose-binding protein (RBP) is exported to the periplasm of Escherichia coli via the general export pathway. An rbsB-lacZ gene fusion was constructed and used to select mutants defective in RBP export. The spontaneous Lac+ mutants isolated in this selection contained either single-amino-acid substitutions or a deletion of the RBP signal sequence. Intact rbsB genes containing eight different point mutations in the signal sequence were reconstructed, and the effects of the mutations on RBP export were examined. Most of the mutations caused severe defects in RBP export. In addition, different suppressor mutations in SecY/PrlA protein were analyzed for their effects on the export of RBP signal sequence mutants in the presence or absence of SecB. Several RBP signal sequence mutants were efficiently suppressed, but others were not suppressed. Export of an RBP signal sequence mutant in prlA mutant strains was partially dependent on SecB, which is in contrast to the SecB independence of wild-type RBP export. However, the kinetics of export of an RBP signal sequence mutant point to a rapid loss of pre-RBP export competence, which occurs in strains containing or lacking SecB. These results suggest that SecB does not stabilize the export-competent conformation of RBP and may affect translocation by stabilizing the binding of pre-RBP at the translocation site.     

A signal sequence is not required for protein export in prlA mutants of Escherichia coli.

The prlA/secY gene, which codes for an integral membrane protein component of the Escherichia coli protein export machinery, is the locus of the strongest suppressors of signal sequence mutations. We demonstrate that two exported proteins of E.coli, maltose-binding protein and alkaline phosphatase, each lacking its entire signal sequence, are exported to the periplasm in several prlA mutants. The export efficiency can be substantial; in a strain carrying the prlA4 allele, 30% of signal-sequenceless alkaline phosphatase is exported to the periplasm. Other components of the E.coli export machinery, including SecA, are required for this export. SecB is required for the export of signal-sequenceless alkaline phosphatase even though the normal export of alkaline phosphatase does not require this chaperonin. Our findings indicate that signal sequences confer speed and efficiency upon the export process, but that they are not always essential for export. Entry into the export pathway may involve components that so overlap in function that the absence of a signal sequence can be compensated for, or there may exist one or more means of entry that do not require signal sequences at all.     

Intragenic reversion mutations that improve export of maltose-binding protein in Escherichia coli malE signal sequence mutants

Escherichia coli strains harboring malE signal sequence point mutations accumulate export-defective precursor maltose-binding protein (MBP) in the cytoplasm. Beginning with these mutants, a number of spontaneous intragenic revertants have been obtained in which export of the MBP to the periplasm is either partially or totally restored. With a single exception, each of the reversion mutations resulted in an increase in the overall hydrophobicity of the signal peptide hydrophobic core by one of five different mechanisms. In some revertants, MBP export was achieved at a rate comparable to the wild type MBP; in other cases, the rate of MBP export was significantly slower than wild type. The results indicate that the overall hydrophobicity of the signal peptide, rather than the absolute length of its uninterrupted hydrophobic core, is a major determinant of MBP export competency. An alteration at residue 19 of the mature MBP also has been identified that provides fairly efficient suppression of the export defect in the adjacent signal peptide, further suggesting that important export information may reside in this region of the precursor protein.     

Molecular components of the signal sequence that function in the initiation of protein export

We are studying the mechanism by which the LamB protein is exported to the outer membrane of Escherichia coli. Using two selection procedures based on gene fusions, we have identified a number of mutations that cause alterations in the LamB signal sequence. Characterization of the mutant strains revealed that although many such mutations block LamB export to greater than 95%, others have essentially no effect. These results allow an analysis of the functions performed by the various molecular components of the signal sequence. Our results suggest that a critical subset of four amino acids is contained within the central hydrophobic core of the LamB signal sequence. If this core can assume an alpha-helical conformation, these four amino acids comprise a recognition site that interacts with a component of the cellular export machinery. Since mechanisms of protein localization appear to have been conserved during evolution, the principles established by these results should be applicable to similar studies in eukaryotic cells.     

Heat shock protein 25 plays multiple roles during mouse skin development

Heat shock protein (Hsp) 25 is a member of the small Hsp family. High levels of Hsp25 can be detected in skin. During adult epidermis differentiation, the concentration of Hsp25 increases as the distance of keratinocytes from the basal layer increases, in parallel with the extent of keratinization. We previously showed that Hsp25, mouse keratin (MK) 5, and MK14 participated in the formation of characteristic ring-shaped aggregates during the differentiation of the PAM212 keratinocyte cell line. We suggested that Hsp25 was involved in the disorganization of the MK5-MK14 keratin network before the establishment of the MK1-MK10 keratin network at the beginning of epidermis stratification. In this study, we have investigated the distribution of Hsp25 and keratins throughout skin development. We demonstrate that the distribution of Hsp25 and MK5 in the epidermis at the beginning of stratification and before keratinization is similar to that observed in PAM212 keratinocytes. These results indicate that there is a strong correlation between the mechanism we described ex vivo and the events taking place in vivo. Moreover, we show that Hsp25 is produced in different cell types in the epidermis and in the hair follicle at different stages of their development. Thus, our results suggest that Hsp25 is involved in more than one process during skin development.     

Biophysical studies of signal peptides: Implications for signal sequence functions and the involvement of lipid in protein export

This review discusses efforts to understand the mode of action of signal sequences by biophysical study of synthetic peptides corresponding to these protein localization signals. On the basis of reports from several laboratories, it is now clear that signal peptides may adopt a variety of conformations, depending on their local environment. In membrane-mimetic systems like detergent micelles or lipid vesicles, they have a high tendency to form helices. Ability to take up a helical conformation appears to be required at some point in the function of a signal sequence, since some peptides corresponding to export-defective signal sequences display reduced helical potential. By contrast, functional signal sequences share a high capacity to adopt helices. High affinity for organized lipid assemblies, like monolayers or vesicles, is also a property of functional signal sequences. This correlation suggests a role for direct interaction of signal sequences with the lipids of the cytoplasmic membranein vivo. Supporting this role are studies of the influence of signal peptides on lipid structure, which reveal an ability of these peptides to pertub lipid packing and to alter the phase state of the lipids. Insertion of the signal sequencein vivo could substantially reduce the barrier for translocation of the mature chain. Lastly, synthetic signal peptides have been added to native membranes and found to inhibit translocation of precursor proteins. This approach bridges the biophysical and the biochemical aspects of protein export and promises to shed light on the functional correlates of the properties and interactions observed in model systems.     

Suppressor mutations that restore export of a protein with a defective signal sequence

A selection procedure is described that should allow the genetic identification of cellular components involved in the process of protein localization in Escherichia coli. This procedure makes use of mutations that alter the signal sequence of the λ receptor protein (product of the lamB gene), and prevent export of this protein to its normal outer membrane location. Several suppressor mutations have been identified that restore export of the mutant λ receptor protein. Mapping experiments show that the suppressor phenotype is the result of mutations in any of at least three different chromosomal loci. One class of suppressor mutations, the class containing the largest number of independent isolates, maps in the major ribosomal gene cluster, suggesting that the suppressor phenotype is the consequence of an altered ribosomal protein. This class of suppressors phenotypically suppresses all known export-defective mutations, internal to the signal sequence region of the lamB gene. These results suggest that ribosomes play an important role in the export of λ receptor to the outer membrane.     

Sequence analysis suggests that tetra-nucleotides signal the termination of protein synthesis in eukaryotes.

An increasing number of cases where tri-nucleotide stop codons do not signal the termination of protein synthesis are being reported. In order to identify what constitutes an efficient stop signal, we analysed the region around natural stop codons in genes from a wide variety of eukaryotic species and gene families. Certain stop codons and nucleotides following stop codons are over-represented, and this pattern is accentuated in highly expressed genes. For example, the preferred signal for Saccharomyces cerevisiae and Drosophila melanogaster highly expressed genes is UAAG, and generally the signals UAA(A/G) and UGA(A/G) are preferred in eukaryotes. The GC% of the organism or DNA region can affect whether there is A or G in the second or fourth positions. We suggest therefore, that the stop codon and the nucleotide following it comprise a tetra-nucleotide stop signal. A model is proposed in which the polypeptide chain release factor, a protein, recognises this sequence, but will tolerate some substitution, particularly A to G in the second or third positions.     

Generation of influenza A virus NS2 (NEP) mutants with an altered nuclear export signal sequence

The NS2 (NEP) protein of influenza A virus contains a highly conserved nuclear export signal (NES) motif in its amino-terminal region (12ILMRMSKMQL21, A/WSN/33), which is thought to be required for nuclear export of viral ribonucleoprotein complexes (vRNPs) mediated by a cellular export factor, CRM1. However, simultaneous replacement of three hydrophobic residues in the NES with alanine does not affect NS2 (NEP) binding to CRM1, although the virus with these mutations is not viable. To determine the extent of sequence conservation required by the NS2 (NEP) NES for its export function during viral replication, we randomly introduced mutations by degenerative mutagenesis into the region of NS cDNA encoding the NS2 (NEP) NES and then attempted to generate mutant viruses containing these alterations by reverse genetics. Sequence analysis of the recovered viruses showed that although some of the mutants possessed amino acids other than those conserved in the NES, hydrophobicity within this motif was maintained. Nuclear export of vRNPs representing all of the mutant viruses was completely inhibited in the presence of a CRM1 inhibitor, leptomycin B, as was the transport of wild-type virus, indicating that the CRM1-mediated pathway is responsible for the nuclear export of both wild-type and mutant vRNPs. The vRNPs of some of the mutant viruses were exported in a delayed manner, resulting in limited viral growth in cell culture and in mice. These results suggest that the NES motif may be an attractive target for the introduction of attenuating mutations in the production of live vaccine viruses.     

The roles of signal peptide and mature protein in RNase (barnase) export from Bacillus subtilis

Barnase, an extracellular RNAse from Bacillus amyloliquefaciens is secreted post-translationally from B. subtilis. The rate of secretion of barnase from B. subtilis was improved by replacement of the barnase signal peptide with a heterologous signal peptide. However, the barnase signal peptide exported Escherichia coli alkaline phosphatase faster than mature barnase. Heat shock of B. subtilis cells did not significantly alter the export of barnase using the barnase signal peptide. The slow rate of export of barnase from B. subtilis is due to both the signal peptide and the mature protein sequence rather than either alone.     

Kinesin-1 plays multiple roles during the vaccinia virus life cycle

The cytoplasmic distribution of cellular structures is known to depend on the balance between plus- and minus-end-directed motor complexes. Among the plus-end-directed kinesins, kinesin-1 and -2 have been implicated in the outward movement of many organelles. To test for a role of kinesin-1 previous studies mostly relied on the overexpression of dominant-negative kinesin-1 constructs. The latter are often cytotoxic, modify the microtubule network and indirect effects related to altered microtubule dynamics should be excluded. In the present study we present a novel kinesin-1 construct, encompassing the first 330 amino acids of kinesin heavy chain fused to GFP (kin330-GFP) that does not alter microtubules upon its overexpression. Kin330-GFP functionally inhibits kinesin-1 because it induces the peri-nuclear accumulation of mitochondria and intermediate filaments. Using this construct and previously established siRNA-mediated knock-down of kinesin-2 function, we assess the role of both motors in the subcellular distribution of distinct steps of the vaccinia virus (VV) life cycle. We show that kinesin-1, but not kinesin-2, contributes to the specific cytoplasmic distribution of three of the four steps of VV morphogenesis tested. These results are discussed with respect to the possible regulation of kinesin-1 during VV infection.     

Both transmembrane domains of SecG contribute to signal sequence recognition by the Escherichia coli protein export machinery

A chimeric protein containing the uncleaved signal sequence of plasminogen activators inhibitor-2 (PAI2) fused to alkaline phosphatase (AP) interferes with Escherichia coli protein export and arrests growth. Suppressors of this toxicity include secG mutations that define the Thr-41-Leu-42-Phe-43 (TLF) domain of SecG. These mutations slow down the export of PAI2-AP. Another construct encoding a truncated PAI2 signal sequence (hB-AP) is also toxic. Most suppressors exert their effect on both chimeric proteins. We describe here five secG suppressors that only suppress the toxicity of hB-AP and selectively slow down its export. These mutations do not alter the TLF domain: three encode truncated SecG, whereas two introduce Arg residues in the transmembrane domains of SecG. The shortest truncated protein only contains 13 residues of SecG, suggesting that the mutation is equivalent to a null allele. Indeed, a secG disruption selectively suppresses the toxicity of hB-AP. However, the missense mutations are not null alleles. They allow SecG binding to SecYE, although with reduced affinity. Furthermore, these mutated SecG are functional, as they facilitate the export of endogenous proteins. Thus, SecG participates in signal sequence recognition, and both transmembrane domains of SecG contribute to ensure normal signal sequence recognition by the translocase.     

Ethylene plays multiple nonprimary roles in modulating the gravitropic response in tomato.

Ethylene is known to interact with auxin in regulating stem growth, and yet evidence for the role of ethylene in tropic responses is contradictory. Our analysis of four mutants of tomato (Lycopersicon esculentum) altered in their response to gravity, auxin, and/or ethylene revealed concentration-dependent modulation of shoot gravitropism by ethylene. Ethylene inhibitors reduce wild-type gravicurvature, and extremely low (0.0005-0.001 microliter L-1) ethylene concentrations can restore the reduced gravitropic response of the auxin-resistant dgt (diageotropica) mutant to wild-type levels. Slightly higher concentrations of ethylene inhibit the gravitropic response of all but the ethylene-insensitive nr (never-ripe) mutant. The gravitropic responses of nr and the constitutive-response mutant epi (epinastic) are slightly and significantly delayed, respectively, but otherwise normal. The reversal of shoot gravicurvature by red light in the lz-2 (lazy-2) mutant is not affected by ethylene. Taken together, these data indicate that, although ethylene does not play a primary role in the gravitropic response of tomato, low levels of ethylene are necessary for a full gravitropic response, and moderate levels of the hormone specifically inhibit gravicurvature in a manner different from ethylene inhibition of overall growth.     

UDP-glucose dehydrogenase plays multiple roles in the biology of the pathogenic fungus Cryptococcus neoformans

Cryptococcus neoformans is a pathogenic fungus surrounded by an elaborate polysaccharide capsule that is strictly required for its virulence in humans and other mammals. Nearly half of the sugar residues in the capsule are derived from UDP-glucuronic acid or its metabolites. To examine the role of these nucleotide sugars in C. neoformans, the gene encoding UDP-glucose dehydrogenase was disrupted. Mass spectrometry analysis of nucleotide sugar pools showed that the resulting mutant lacked both UDP-glucuronic acid and its downstream product, UDP-xylose, thus confirming the effect of the knockout and indicating that an alternate pathway for UDP-glucuronic acid production was not used. The mutant was dramatically affected by the lack of specific sugar donors, demonstrating altered cell integrity, temperature sensitivity, lack of growth in an animal model of cryptococcosis, and morphological defects. Additionally, the polysaccharide capsule could not be detected on the mutant cells, although the possibility remains that abbreviated forms of capsule components are made, possibly without proper surface display. The capsule defect is largely independent of the other observed changes, as cells that are acapsular because of mutations in other genes show lack of virulence but do not exhibit alterations in cell integrity, temperature sensitivity, or cellular morphology. All of the observed alterations were reversed by correction of the gene disruption.     

Nuclear import of hepatic glucokinase depends upon glucokinase regulatory protein, whereas export is due to a nuclear export signal sequence in glucokinase

Hepatic glucokinase (GK) moves between the nucleus and cytoplasm in response to metabolic alterations. Here, using heterologous cell systems, we have found that at least two different mechanisms are involved in the intracellular movement of GK. In the absence of the GK regulatory protein (GKRP) GK resides only in the cytoplasm. However, in the presence of GKRP, GK moves to the nucleus and resides there in association with this protein until changes in the metabolic milieu prompt its release. GK does not contain a nuclear localization signal sequence and does not enter the nucleus in a GKRP-independent manner because cells treated with leptomycin B, a specific inhibitor of leucine-rich NES-dependent nuclear export, do not accumulate GK in the nucleus. Instead, entry of GK into the nucleus appears to occur via a piggy-back mechanism that involves binding to GKRP. Nuclear export of GK, which occurs after its release from GKRP, is due to a leucine-rich nuclear export signal within the protein ((300)ELVRLVLLKLV(310)). Thus, GKRP appears to function as both a nuclear chaperone and metabolic sensor and is a critical component of a hepatic GK translocation cycle for regulating the activity of this enzyme in response to metabolic alterations.     

Further sequence analysis of the phage lambda receptor site. Possible implications for the organization of the lamB protein in Escherichia coli K12

We present the DNA sequence alterations due to seven lamB missense mutations yielding resistance to phages lambda and K10. They reveal five different amino acid positions in the LamB protein. Three positions (245, 247 and 249) define a new region required for phage adsorption. The two other positions (148 and 152) belong to a region where mutations to phage resistance has already been detected. These two regions are hydrophilic and could belong to turns of the protein located at the surface of the cell. All the missense mutational alterations to phage resistance sequenced in the LamB protein correspond to 10 sites located in four different segments of the polypeptide chain. We discuss their location in terms of the notion of phage receptor site and of a working model for the organization of this protein in the outer membrane of Escherichia coli.     

Synthesis and export of the outer membrane lipoprotein in Escherichia coli mutants defective in generalized protein export.

Export of the outer membrane lipoprotein in Escherichia coli was examined in conditionally lethal mutants that were defective in protein export in general, including secA, secB, secC, and secD. Lipoprotein export was affected in a secA(Ts) mutant of E. coli at the nonpermissive temperature; it was also affected in a secA(Am) mutant of E. coli at the permissive temperature, but not at the nonpermissive temperature. The export of lipoprotein occurred normally in E. coli carrying a null secB::Tn5 mutation; on the other hand, the export of an OmpF::Lpp hybrid protein, consisting of the signal sequence plus 11 amino acid residues of mature OmpF and mature lipoprotein, was affected by the secB mutation. The synthesis of lipoprotein was reduced in the secC mutant at the nonpermissive temperature, as was the case for synthesis of the maltose-binding protein, while the synthesis of OmpA was not affected. Lipoprotein export was found to be slightly affected in secD(Cs) mutants at the nonpermissive temperature. These results taken together indicate that the export of lipoprotein shares the common requirements for functional SecA and SecD proteins with other exported proteins, but does not require a functional SecB protein. SecC protein (ribosomal protein S15) is required for the optimal synthesis of lipoprotein.