ABSTRACT
The translocation t(12;22)(p13;q11) creates an MN1-TEL fusion gene leading to acute myeloid leukemia. MN1 is a transcription coactivator of the retinoic acid and vitamin D receptors, and TEL (ETV6) is a member of the E26-transformation-specific family of transcription factors. In MN1-TEL, the transactivating domains of MN1 are combined with the DNA-binding domain of TEL. We show that MN1-TEL inhibits retinoic acid receptor (RAR)-mediated transcription, counteracts coactivators such as p160 and p300, and acts as a dominant-negative mutant of MN1. Compared to MN1, the same transactivation domains in MN1-TEL are poorly stimulated by p160, p300 or histone deacetylase inhibitors, indicating that the block of RAR-mediated transcription by MN1-TEL is caused by dysfunctional transactivation domains rather than by recruitment of corepressors. The mechanism leading to myeloid leukemia in t(12;22) thus differs from the translocations that involve RAR itself.
Subject(s)
Carcinoma, Hepatocellular/pathology , Oncogene Proteins, Fusion/physiology , Receptors, Retinoic Acid/genetics , Retinoid X Receptors/genetics , Transcription Factors/physiology , Transcription, Genetic , Carcinoma, Hepatocellular/genetics , Chromosomes, Human, Pair 12/genetics , Chromosomes, Human, Pair 22/genetics , DNA-Binding Proteins , E1A-Associated p300 Protein/physiology , Enzyme Inhibitors , Genes, Dominant , Histone Deacetylase Inhibitors , Humans , Liver Neoplasms/genetics , Liver Neoplasms/pathology , Mutation , Nuclear Proteins/physiology , Nucleocytoplasmic Transport Proteins/physiology , RNA-Binding Proteins , Reverse Transcriptase Polymerase Chain Reaction , Transcriptional Activation , Transfection , Translocation, Genetic , Tumor Cells, CulturedABSTRACT
In contrast to Gram-negative bacteria, secretory proteins of Gram-positive bacteria only need to traverse a single membrane to enter the extracellular environment. For this reason, Gram-positive bacteria (e.g. various Bacillus species) are often used in industry for the commercial production of extracellular proteins that can be produced in yields of several grams per liter culture medium. The central components of the main protein translocation system (Sec system) of Gram-negative and Gram-positive bacteria show a high degree of conservation, suggesting similar functions and working mechanisms. Despite this fact, several differences can be identified such as the absence of a clear homolog of the secretion-specific chaperone SecB in Gram-positive bacteria. The now available detailed insight into the organization of the Gram-positive protein secretion system and how it differs from the well-characterized system of Escherichia coli may in the future facilitate the exploitation of these organisms in the high level production of heterologous proteins which, so far, is sometimes very inefficient due to one or more bottlenecks in the secretion pathway. In this review, we summarize the current knowledge on the various steps of the protein secretion pathway of Gram-positive bacteria with emphasis on Bacillus subtilis, which during the last decade, has arisen as a model system for the study of protein secretion in this industrially important class of microorganisms.
Subject(s)
Bacterial Proteins/metabolism , Cell Membrane/metabolism , Gram-Positive Bacteria/cytology , Gram-Positive Bacteria/metabolism , Bacillus subtilis/cytology , Bacillus subtilis/genetics , Bacillus subtilis/metabolism , Bacterial Proteins/chemistry , Bacterial Proteins/genetics , Carrier Proteins/metabolism , Cell Wall/metabolism , Gram-Positive Bacteria/genetics , Protein Sorting Signals/genetics , Protein Sorting Signals/physiology , Protein Transport , Signal Recognition Particle/metabolismABSTRACT
CsaA from the Gram-positive bacterium Bacillus subtilis has been identified previously as a suppressor of the growth and protein-export defect of Escherichia coli secA(Ts) mutants. CsaA has chaperone-like activities in vivo and in vitro. To examine the role of CsaA in protein export in B. subtilis, expression of the csaA gene was repressed. While export of most proteins remained unaffected, export of at least two proteins was significantly reduced upon CsaA depletion. CsaA co-immunoprecipitates and co-purifies with the SecA proteins of E. coli and B. subtilis, and binds the B. subtilis preprotein prePhoB. Purified CsaA stimulates the translocation of prePhoB into E. coli membrane vesicles bearing the B. subtilis translocase, whereas it interferes with the SecB-mediated translocation of proOmpA into membrane vesicles of E. coli. The specific interaction with the SecA translocation ATPase and preproteins suggests that CsaA acts as a chaperone that promotes the export of a subset of preproteins in B. subtilis.
Subject(s)
Adenosine Triphosphatases/metabolism , Bacillus subtilis/metabolism , Bacterial Proteins/metabolism , Carrier Proteins/metabolism , Escherichia coli Proteins , Membrane Transport Proteins , Molecular Chaperones , Protein Precursors/metabolism , Adenosine Triphosphatases/isolation & purification , Bacillus subtilis/genetics , Bacterial Proteins/genetics , Bacterial Proteins/isolation & purification , Carrier Proteins/isolation & purification , DNA-Binding Proteins/isolation & purification , DNA-Binding Proteins/metabolism , Escherichia coli/genetics , Escherichia coli/metabolism , Genotype , Phenotype , Plasmids , Protein Binding , Protein Precursors/isolation & purification , Recombinant Proteins/isolation & purification , Recombinant Proteins/metabolism , SEC Translocation Channels , SecA ProteinsABSTRACT
Bacterial protein translocation is mediated by translocase, a multisubunit membrane protein complex that consists of a peripheral ATPase SecA and a preprotein-conducting channel with SecY, SecE, and SecG as subunits. Like Escherichia coli SecG, the Bacillus subtilis homologue, YvaL, dramatically stimulated the ATP-dependent translocation of precursor PhoB (prePhoB) by the B. subtilis SecA-SecYE complex. To systematically determine the functional exchangeability of translocase subunits, all of the relevant combinations of the E. coli and B. subtilis secY, secE, and secG genes were expressed in E. coli. Hybrid SecYEG complexes were overexpressed at high levels. Since SecY could not be overproduced without SecE, these data indicate a stable interaction between the heterologous SecY and SecE subunits. E. coli SecA, but not B. subtilis SecA, supported efficient ATP-dependent translocation of the E. coli precursor OmpA (proOmpA) into inner membrane vesicles containing the hybrid SecYEG complexes, if E. coli SecY and either E. coli SecE or E. coli SecG were present. Translocation of B. subtilis prePhoB, on the other hand, showed a strict dependence on the translocase subunit composition and occurred efficiently only with the homologous translocase. In contrast to E. coli SecA, B. subtilis SecA binds the SecYEG complexes only with low affinity. These results suggest that each translocase subunit contributes in an exclusive manner to the specificity and functionality of the complex.
Subject(s)
Adenosine Triphosphatases/genetics , Adenosine Triphosphatases/metabolism , Bacillus subtilis/enzymology , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Escherichia coli Proteins , Escherichia coli/enzymology , Membrane Transport Proteins , Adenosine Triphosphatases/chemistry , Bacillus subtilis/genetics , Bacillus subtilis/metabolism , Bacterial Proteins/chemistry , Biological Transport , Escherichia coli/genetics , Escherichia coli/metabolism , Protein Multimerization , Protein Precursors/metabolism , Recombinant Fusion Proteins/chemistry , Recombinant Fusion Proteins/metabolism , SEC Translocation Channels , SecA ProteinsABSTRACT
SecA is the dissociatable nucleotide and preprotein binding subunit of the bacterial translocase. The thermodynamics of nucleotide binding to soluble SecA at nucleotide binding site I were determined by isothermal titration calorimetry. Binding of ADP and non-hydrolyzable ATPgammaS is enthalpy-driven (DeltaH(0) of -14.44 and -5.56 kcal/mol, respectively), but is accompanied by opposite entropic contributions (DeltaS(0) of -18.25 and 9.55 cal/mol/K, respectively). ADP binding results in a large change in the heat capacity of SecA (DeltaC(p)=-780 cal/mol/K). It is suggested that ADP binding promotes the interaction between the two thermodynamically discernible domains of SecA which is accompanied by a shielding of hydrophobic surface from solvent.
Subject(s)
Adenosine Diphosphate/metabolism , Adenosine Triphosphatases/metabolism , Adenosine Triphosphate/metabolism , Bacillus subtilis/enzymology , Bacterial Proteins/metabolism , Enzyme Precursors/metabolism , Escherichia coli Proteins , Membrane Transport Proteins , Peptide Fragments/metabolism , Adenosine Diphosphate/chemistry , Adenosine Triphosphatases/chemistry , Adenosine Triphosphate/analogs & derivatives , Adenosine Triphosphate/chemistry , Bacterial Proteins/chemistry , Binding Sites , Biological Transport , Calorimetry, Differential Scanning , Enzyme Precursors/chemistry , Models, Chemical , Peptide Fragments/chemistry , Protein Conformation , SEC Translocation Channels , SecA Proteins , ThermodynamicsABSTRACT
Protein export in Escherichia coli is mediated by translocase, a multisubunit membrane protein complex with SecA as the peripheral subunit and the SecY, SecE, and SecG proteins as the integral membrane domain. In the gram-positive bacterium Bacillus subtilis, SecA, SecY, and SecE have been identified through genetic analysis. Sequence comparison of the Bacillus chromosome identified a potential homologue of SecG, termed YvaL. A chromosomal disruption of the yvaL gene results in mild cold sensitivity and causes a beta-lactamase secretion defect. The cold sensitivity is exacerbated by overexpression of the secretory protein alpha-amylase, whereas growth and beta-lactamase secretion are restored by coexpression of yvaL or the E. coli secG gene. These results indicate that the yvaL gene codes for a protein that is functionally homologous to SecG.
Subject(s)
Bacillus subtilis/genetics , Bacterial Proteins/genetics , Escherichia coli Proteins , Genes, Bacterial , Membrane Proteins/genetics , Amino Acid Sequence , Bacillus subtilis/growth & development , Bacillus subtilis/metabolism , Bacterial Proteins/metabolism , Base Sequence , Biological Transport, Active , Cold Temperature , DNA Primers/genetics , Escherichia coli/genetics , Gene Deletion , Genetic Complementation Test , Molecular Sequence Data , Phenotype , SEC Translocation Channels , Sequence Homology, Amino Acid , Species SpecificityABSTRACT
Bacilli vigorously secrete proteins into the extracellular environment, and are therefore used in industry for the bulk production of enzymes such as proteinases and amylases. Studies on the mechanism of protein translocation in these Gram-positive bacteria have been hampered by the lack of an in vitro system. To establish such a system for Bacillus subtilis, everted membranes were isolated from a strain deficient in the alkaline and neutral protease. Translocation-competent membrane vesicles were obtained only when a broad range proteinase-inhibitor cocktail was used during membrane isolation. This method efficiently prevented proteolysis of SecY, one of the core integral membrane components of the preprotein translocase. Translocation of the urea-denatured precursor of the Bacillus licheniformis alpha-amylase, preAmyL, and B. subtilis alkaline phosphatase, prePhoB, into the B. subtilis membrane vesicles require the B. subtilis SecA protein and are driven by ATP hydrolysis and the proton-motive force. These studies establish an authentic in vitro translocation system for protein secretion in B. subtilis.