D. Oliver - Academia.edu (original) (raw)

Papers by D. Oliver

Research paper thumbnail of Distinct Membrane Binding Properties of N- and C-terminal Domains of Escherichia coli SecA ATPase

Journal of Biological Chemistry, 2000

SecA is a motor protein that drives protein translocation at the Escherichia coli translocon. Sec... more SecA is a motor protein that drives protein translocation at the Escherichia coli translocon. SecA membrane binding has been shown to occur with high affinity at SecYE and low affinity at anionic phospholipids. To dissect SecA-membrane interaction with reference to SecA structure, the membrane binding properties of N-and C-terminal SecA domains, denoted SecA-N664 and SecA-619C, respectively, were characterized. Remarkably, only SecA-N664 bound to the membrane with high affinity, whereas SecA-619C bound with low affinity in a nonsaturable manner through partitioning with phospholipids. Moreover, SecA-N664 and SecA-619C associated with each other to reconstitute wild type binding affinity. Corroborative results were also obtained from membrane binding competition and subcellular fractionation studies along with binding studies to membranes prepared from strains overproducing SecYE protein. Together, these findings indicate that the specific interaction of SecA with SecYE occurs through its Nterminal domain and that the C-terminal domain, although important in SecA membrane cycling at a later stage of translocation, appears to initially assist SecA membrane binding by interaction with phospholipids. These results provide the first evidence for distinct membrane binding characteristics of the two SecA primary domains and their importance for optimal binding activity, and they are significant for understanding SecA dynamics at the translocon.

Research paper thumbnail of Topology of the Integral Membrane Form of Escherichia coli SecA Protein Reveals Multiple Periplasmically Exposed Regions and Modulation by ATP Binding

Journal of Biological Chemistry, 1997

SecA insertion and integration into the Escherichia coli inner membrane is a critical step for th... more SecA insertion and integration into the Escherichia coli inner membrane is a critical step for the catalysis of protein translocation across this layer. To understand this step further, SecA topology was investigated. To determine which regions of SecA are periplasmically exposed, right-side out membrane vesicles were prepared from strains synthesizing monocysteine SecA variants produced by mutagenesis and probed with a membrane-impermeant sulfhydryl-labeling reagent. To determine which regions of SecA contain membraneintegration determinants, inverted inner membrane vesicles were subjected to proteolysis, and integralmembrane fragments of SecA were identified with region-specific antibodies. The membrane association properties of various truncated SecA species produced in vivo were also determined. Our analysis indicates that the membrane topology of SecA is complex with amino-terminal, central, and carboxyl-terminal regions of SecA integrated into the membrane where portions are periplasmically accessible. Furthermore, the insertion and penetration of the amino-terminal third of SecA, which includes the proposed preprotein-binding domain, is subject to modulation by ATP binding. The importance of these studies to the cycle of membrane insertion and de-insertion of SecA that promotes protein translocation and SecA's proximity to the preprotein channel are discussed.

Research paper thumbnail of Regulation of Escherichia coli secA by cellular protein secretion proficiency requires an intact gene X signal sequence and an active translocon

Journal of bacteriology, 1998

secA is translationally regulated by the protein secretion proficiency state of the Escherichia c... more secA is translationally regulated by the protein secretion proficiency state of the Escherichia coli cell. This regulation was explored by making signal sequence mutations in the gene upstream of secA, gene X, which promotes secA translational coupling. Gene X signal sequence mutants were constitutive for secA expression, while prlA alleles partially restored secA regulation. These results show that interaction of the pre-gene X protein with the translocon is required for proper secA regulation. Furthermore, gene X signal sequence mutations disrupted secA regulation only in the cis configuration. We propose that nascent pre-gene X protein interacts with the translocon during its secretion to constitute the secretion sensor.

Research paper thumbnail of ssaD1, a suppressor of secA51(Ts) that renders growth of Escherichia coli cold sensitive, is an early amber mutation in the transcription factor gene nusB

Journal of bacteriology, 1994

Complementation analysis of the ssaD1 mutation, isolated as a suppressor of the secA51(Ts) mutati... more Complementation analysis of the ssaD1 mutation, isolated as a suppressor of the secA51(Ts) mutation that renders growth of Escherichia coli cold sensitive, was used to show that ssaD corresponds to nusB, a gene known to be important in transcription antitermination. DNA sequence analysis of the ssaD1 allele showed that it creates an amber mutation in the 15th codon of nusB. Analysis of the effect of different levels of NusB protein on secA transcription and translation suggested that NusB plays little or no role in the control of secA expression. Accordingly, mechanisms by which nusB inactivation can lead to suppression of secA51(Ts) and secY24(Ts) mutations without affecting secA expression need to be considered.

Research paper thumbnail of Competition between ribosome and SecA binding promotes Escherichia coli secA translational regulation

RNA (New York, N.Y.), 1995

SecA protein, the protein translocation ATPase of Escherichia coli, autogenously regulates its tr... more SecA protein, the protein translocation ATPase of Escherichia coli, autogenously regulates its translation during normal protein secretion by binding to a secretion-responsive element located near the 5' end of its gene on geneX-secA mRNA. In order to characterize this autoregulation further, RNA footprinting and primerextension inhibition (toeprinting) studies were carried out with a segment of geneX-secA RNA, 30S ribosomal subunits and tRNAfMet along with purified SecA protein. The results show that ribosome and SecA-binding sites overlap, indicating that a simple competition for binding of geneX-secA mRNA presumably governs the translation initiation step. Further analysis showed that SecA protein was able to specifically dissociate a preformed 30S-tRNAfMet-geneX-secA RNA ternary complex as indicated by the disappearance of its characteristic toeprint after SecA addition. These findings are consistent with secA autoregulation, and they suggest a novel mechanism for the autore...

Research paper thumbnail of Mutations that alter the signal sequence of alkaline phosphatase in Escherichia coli

Journal of bacteriology, 1983

A phoA-lacZ gene fusion was used to isolate mutants altered in the alkaline phosphatase signal se... more A phoA-lacZ gene fusion was used to isolate mutants altered in the alkaline phosphatase signal sequence. This was done by selecting Lac+ mutants from a phoA-lacZ fusion strain that produces a membrane-bound hybrid protein and is unable to grow on lactose. Two such mutant derivatives were characterized. The mutations lie within the phoA portion of the fused gene and cause internalization of the hybrid protein. When the mutations were genetically recombined into an otherwise wild-type phoA gene, they interfered with export of alkaline phosphatase to the periplasm. The mutant alkaline phosphatase protein was found instead in the cytoplasm in precursor form. DNA sequence analysis demonstrated that both mutations lead to amino acid alterations in the signal sequence of alkaline phosphatase.

Research paper thumbnail of Integration of SecA protein into the Escherichia coli inner membrane is regulated by its amino-terminal ATP-binding domain

Molecular Microbiology, 1996

SecA protein, the ATPase promoting translocation of proteins across the Escherichia coli inner me... more SecA protein, the ATPase promoting translocation of proteins across the Escherichia coli inner membrane, contains two ATP-binding domains that differ greatly in their affinity for bound nucleotide. In order to define more precisely the location of the high-affinity nucleotide-binding site, oligonucleotide-directed mutagenesis was used to introduce cysteine residues into the SecA sequence, and a cysteine-specific cleavage reagent was employed to generate defined peptides of SecA protein after photocross-linking with [a-32P]-ATP. This analysis revealed that the nucleotide was crosslinked between amino acid residues 75 and 97 of SecA protein. The biochemical function of the high affinity ATP-binding domain was explored by subcellular fractionation studies which demonstrated that SecA proteins defective in this region were found almost exclusively in their integral membrane form, while SecA proteins with defects in the low-affinity ATPdomain showed a normal distribution of cytosolic, peripheral and integral membrane forms. Interestingly, the SecA5l(Ts) protein that has a Leu to Pro substitution at amino acid residue 43 bound ATP with high affinity, but its fractionation pattern and translocation ATPase activity were similar to those of proteins with defects in the high-affinity ATPbinding site. These results delimit more precisely the high-affinity ATP-binding domain of SecA, indicate the importance of the early amino-terminal region of SecA protein in the functioning of this domain, and demonstrate the role of this domain in regulating penetration of SecA protein into the inner membrane. Our results lead to a simple model for the regulation of a cycle of SecA insertion into, and de-insertion from,

Research paper thumbnail of Identification of a Region of Interaction between Escherichia coli SecA and SecY Proteins

Journal of Biological Chemistry, 1997

SecA ATPase promotes Escherichia coli protein translocation by its association with the preprotei... more SecA ATPase promotes Escherichia coli protein translocation by its association with the preprotein or preprotein-SecB complex, anionic phospholipids, and the other core component of translocase, integral membrane protein SecYEG. Using ligand affinity blotting we demonstrate a direct interaction of SecA with SecY protein. Proteolysis and gene truncation or fusion studies were used to further define this interaction. Our results demonstrate that the carboxyl-terminal third of SecA protein binds to the amino-terminal 107 amino acid residues of SecY protein. The direct demonstration of these interactions culminate studies that have inferred an interaction between SecA and SecYEG, and they are consistent with studies suggesting that this region of SecA interacts with the inner membrane.

Research paper thumbnail of Nucleotide Binding Activity of SecA Homodimer Is Conformationally Regulated by Temperature and Altered by prlD and azi Mutations

Journal of Biological Chemistry, 2000

SecA ATPase is critical for protein translocation across the Escherichia coli inner membrane. To ... more SecA ATPase is critical for protein translocation across the Escherichia coli inner membrane. To understand this activity further, the high affinity nucleotide binding activity of SecA was characterized. We found that at 4°C SecA homodimer binds one ADP molecule with high affinity. This nucleotide binding activity was conformationally regulated by temperature: at low temperature SecA affinity for ADP was high with a slow exchange rate, whereas at high temperature the converse was true. Azi-and PrlD-SecA proteins that confer azide-resistant and signal sequence suppressor phenotypes were found to have reduced affinity for ADP and accelerated exchange rates compared with wild type SecA. Consistent with this observation, fluorescence and proteolysis studies indicated that these proteins had a conformationally relaxed state at a reduced temperature compared with SecA. The level of Azi-and PrlD-SecA protein was also elevated in inverted membrane vesicles where it displayed higher membrane ATPase activity. These results provide the first direct evidence for conformational regulation of the SecA-dependent nucleotide cycle, its alteration in azi and prlD mutants, and its relevance to in vivo protein export.

Research paper thumbnail of Revised Translation Start Site for secM Defines an Atypical Signal Peptide That Regulates Escherichia coli secA Expression

Journal of Bacteriology, 2000

The secretion-responsive regulation of Escherichia coli secA occurs by coupling its translation t... more The secretion-responsive regulation of Escherichia coli secA occurs by coupling its translation to the translation and secretion of an upstream regulator, secM (formerly geneX). We revise the translational start site for secM, defining a new signal peptide sequence with an extended amino-terminal region. Mutational studies indicate that certain atypical amino acyl residues within this extended region are critical for propersecA regulation.

Research paper thumbnail of Re-examination of the Role of the Amino-Terminus of SecA in Promoting Its Dimerization and Functional State

Biophysical Journal, 2009

The SecA nanomotor promotes protein translocation in eubacteria by binding both protein cargo and... more The SecA nanomotor promotes protein translocation in eubacteria by binding both protein cargo and the protein-conducting channel and by undergoing ATP-driven conformation cycles that drive this process. There are conflicting reports about whether SecA functions as a ...

Research paper thumbnail of Protein Secretion in Escherichia Coli

Annual Review of Microbiology, 1985

... 624 OLIVER The Basic Amino-Terminus Oligonucleotide-directed mutagenesis (149) has been used ... more ... 624 OLIVER The Basic Amino-Terminus Oligonucleotide-directed mutagenesis (149) has been used to remove one or both of the lysine residues in the signal sequence of the major outer membrane lipoprotein (Lpp) and replace them with neutral or acidic amino acids (76, 168 ...

Research paper thumbnail of Nucleotide Binding Activity of SecA Homodimer Is Conformationally Regulated by Temperature and Altered by prlD and azi Mutations

Journal of Biological Chemistry, 2000

SecA ATPase is critical for protein translocation across the Escherichia coli inner membrane. To ... more SecA ATPase is critical for protein translocation across the Escherichia coli inner membrane. To understand this activity further, the high affinity nucleotide binding activity of SecA was characterized. We found that at 4°C SecA homodimer binds one ADP molecule with high affinity. This nucleotide binding activity was conformationally regulated by temperature: at low temperature SecA affinity for ADP was high with a slow exchange rate, whereas at high temperature the converse was true. Azi-and PrlD-SecA proteins that confer azide-resistant and signal sequence suppressor phenotypes were found to have reduced affinity for ADP and accelerated exchange rates compared with wild type SecA. Consistent with this observation, fluorescence and proteolysis studies indicated that these proteins had a conformationally relaxed state at a reduced temperature compared with SecA. The level of Azi-and PrlD-SecA protein was also elevated in inverted membrane vesicles where it displayed higher membrane ATPase activity. These results provide the first direct evidence for conformational regulation of the SecA-dependent nucleotide cycle, its alteration in azi and prlD mutants, and its relevance to in vivo protein export.

Research paper thumbnail of The Variable Subdomain of Escherichia coli SecA Functions To Regulate SecA ATPase Activity and ADP Release

Journal of Bacteriology, 2012

Bacterial SecA proteins can be categorized by the presence or absence of a variable subdomain (VA... more Bacterial SecA proteins can be categorized by the presence or absence of a variable subdomain (VAR) located within nucleotidebinding domain II of the SecA DEAD motor. Here we show that VAR is dispensable for SecA function, since the VAR deletion mutant secA⌬519-547 displayed a wild-type rate of cellular growth and protein export. Loss or gain of VAR is extremely rare in the history of bacterial evolution, indicating that it appears to contribute to secA function within the relevant species in their natural environments. VAR removal also results in additional secA phenotypes: azide resistance (Azi r ) and suppression of signal sequence defects (PrlD). The SecA⌬(519 -547) protein was found to be modestly hyperactive for SecA ATPase activities and displayed an accelerated rate of ADP release, consistent with the biochemical basis of azide resistance. Based on our findings, we discuss models whereby VAR allosterically regulates SecA DEAD motor function at SecYEG.

Research paper thumbnail of Distinct Membrane Binding Properties of N- and C-terminal Domains of Escherichia coli SecA ATPase

Journal of Biological Chemistry, 2000

SecA is a motor protein that drives protein translocation at the Escherichia coli translocon. Sec... more SecA is a motor protein that drives protein translocation at the Escherichia coli translocon. SecA membrane binding has been shown to occur with high affinity at SecYE and low affinity at anionic phospholipids. To dissect SecA-membrane interaction with reference to SecA structure, the membrane binding properties of N-and C-terminal SecA domains, denoted SecA-N664 and SecA-619C, respectively, were characterized. Remarkably, only SecA-N664 bound to the membrane with high affinity, whereas SecA-619C bound with low affinity in a nonsaturable manner through partitioning with phospholipids. Moreover, SecA-N664 and SecA-619C associated with each other to reconstitute wild type binding affinity. Corroborative results were also obtained from membrane binding competition and subcellular fractionation studies along with binding studies to membranes prepared from strains overproducing SecYE protein. Together, these findings indicate that the specific interaction of SecA with SecYE occurs through its Nterminal domain and that the C-terminal domain, although important in SecA membrane cycling at a later stage of translocation, appears to initially assist SecA membrane binding by interaction with phospholipids. These results provide the first evidence for distinct membrane binding characteristics of the two SecA primary domains and their importance for optimal binding activity, and they are significant for understanding SecA dynamics at the translocon.

Research paper thumbnail of Topology of the Integral Membrane Form of Escherichia coli SecA Protein Reveals Multiple Periplasmically Exposed Regions and Modulation by ATP Binding

Journal of Biological Chemistry, 1997

SecA insertion and integration into the Escherichia coli inner membrane is a critical step for th... more SecA insertion and integration into the Escherichia coli inner membrane is a critical step for the catalysis of protein translocation across this layer. To understand this step further, SecA topology was investigated. To determine which regions of SecA are periplasmically exposed, right-side out membrane vesicles were prepared from strains synthesizing monocysteine SecA variants produced by mutagenesis and probed with a membrane-impermeant sulfhydryl-labeling reagent. To determine which regions of SecA contain membraneintegration determinants, inverted inner membrane vesicles were subjected to proteolysis, and integralmembrane fragments of SecA were identified with region-specific antibodies. The membrane association properties of various truncated SecA species produced in vivo were also determined. Our analysis indicates that the membrane topology of SecA is complex with amino-terminal, central, and carboxyl-terminal regions of SecA integrated into the membrane where portions are periplasmically accessible. Furthermore, the insertion and penetration of the amino-terminal third of SecA, which includes the proposed preprotein-binding domain, is subject to modulation by ATP binding. The importance of these studies to the cycle of membrane insertion and de-insertion of SecA that promotes protein translocation and SecA's proximity to the preprotein channel are discussed.

Research paper thumbnail of Regulation of Escherichia coli secA by cellular protein secretion proficiency requires an intact gene X signal sequence and an active translocon

Journal of bacteriology, 1998

secA is translationally regulated by the protein secretion proficiency state of the Escherichia c... more secA is translationally regulated by the protein secretion proficiency state of the Escherichia coli cell. This regulation was explored by making signal sequence mutations in the gene upstream of secA, gene X, which promotes secA translational coupling. Gene X signal sequence mutants were constitutive for secA expression, while prlA alleles partially restored secA regulation. These results show that interaction of the pre-gene X protein with the translocon is required for proper secA regulation. Furthermore, gene X signal sequence mutations disrupted secA regulation only in the cis configuration. We propose that nascent pre-gene X protein interacts with the translocon during its secretion to constitute the secretion sensor.

Research paper thumbnail of ssaD1, a suppressor of secA51(Ts) that renders growth of Escherichia coli cold sensitive, is an early amber mutation in the transcription factor gene nusB

Journal of bacteriology, 1994

Complementation analysis of the ssaD1 mutation, isolated as a suppressor of the secA51(Ts) mutati... more Complementation analysis of the ssaD1 mutation, isolated as a suppressor of the secA51(Ts) mutation that renders growth of Escherichia coli cold sensitive, was used to show that ssaD corresponds to nusB, a gene known to be important in transcription antitermination. DNA sequence analysis of the ssaD1 allele showed that it creates an amber mutation in the 15th codon of nusB. Analysis of the effect of different levels of NusB protein on secA transcription and translation suggested that NusB plays little or no role in the control of secA expression. Accordingly, mechanisms by which nusB inactivation can lead to suppression of secA51(Ts) and secY24(Ts) mutations without affecting secA expression need to be considered.

Research paper thumbnail of Competition between ribosome and SecA binding promotes Escherichia coli secA translational regulation

RNA (New York, N.Y.), 1995

SecA protein, the protein translocation ATPase of Escherichia coli, autogenously regulates its tr... more SecA protein, the protein translocation ATPase of Escherichia coli, autogenously regulates its translation during normal protein secretion by binding to a secretion-responsive element located near the 5' end of its gene on geneX-secA mRNA. In order to characterize this autoregulation further, RNA footprinting and primerextension inhibition (toeprinting) studies were carried out with a segment of geneX-secA RNA, 30S ribosomal subunits and tRNAfMet along with purified SecA protein. The results show that ribosome and SecA-binding sites overlap, indicating that a simple competition for binding of geneX-secA mRNA presumably governs the translation initiation step. Further analysis showed that SecA protein was able to specifically dissociate a preformed 30S-tRNAfMet-geneX-secA RNA ternary complex as indicated by the disappearance of its characteristic toeprint after SecA addition. These findings are consistent with secA autoregulation, and they suggest a novel mechanism for the autore...

Research paper thumbnail of Mutations that alter the signal sequence of alkaline phosphatase in Escherichia coli

Journal of bacteriology, 1983

A phoA-lacZ gene fusion was used to isolate mutants altered in the alkaline phosphatase signal se... more A phoA-lacZ gene fusion was used to isolate mutants altered in the alkaline phosphatase signal sequence. This was done by selecting Lac+ mutants from a phoA-lacZ fusion strain that produces a membrane-bound hybrid protein and is unable to grow on lactose. Two such mutant derivatives were characterized. The mutations lie within the phoA portion of the fused gene and cause internalization of the hybrid protein. When the mutations were genetically recombined into an otherwise wild-type phoA gene, they interfered with export of alkaline phosphatase to the periplasm. The mutant alkaline phosphatase protein was found instead in the cytoplasm in precursor form. DNA sequence analysis demonstrated that both mutations lead to amino acid alterations in the signal sequence of alkaline phosphatase.

Research paper thumbnail of Integration of SecA protein into the Escherichia coli inner membrane is regulated by its amino-terminal ATP-binding domain

Molecular Microbiology, 1996

SecA protein, the ATPase promoting translocation of proteins across the Escherichia coli inner me... more SecA protein, the ATPase promoting translocation of proteins across the Escherichia coli inner membrane, contains two ATP-binding domains that differ greatly in their affinity for bound nucleotide. In order to define more precisely the location of the high-affinity nucleotide-binding site, oligonucleotide-directed mutagenesis was used to introduce cysteine residues into the SecA sequence, and a cysteine-specific cleavage reagent was employed to generate defined peptides of SecA protein after photocross-linking with [a-32P]-ATP. This analysis revealed that the nucleotide was crosslinked between amino acid residues 75 and 97 of SecA protein. The biochemical function of the high affinity ATP-binding domain was explored by subcellular fractionation studies which demonstrated that SecA proteins defective in this region were found almost exclusively in their integral membrane form, while SecA proteins with defects in the low-affinity ATPdomain showed a normal distribution of cytosolic, peripheral and integral membrane forms. Interestingly, the SecA5l(Ts) protein that has a Leu to Pro substitution at amino acid residue 43 bound ATP with high affinity, but its fractionation pattern and translocation ATPase activity were similar to those of proteins with defects in the high-affinity ATPbinding site. These results delimit more precisely the high-affinity ATP-binding domain of SecA, indicate the importance of the early amino-terminal region of SecA protein in the functioning of this domain, and demonstrate the role of this domain in regulating penetration of SecA protein into the inner membrane. Our results lead to a simple model for the regulation of a cycle of SecA insertion into, and de-insertion from,

Research paper thumbnail of Identification of a Region of Interaction between Escherichia coli SecA and SecY Proteins

Journal of Biological Chemistry, 1997

SecA ATPase promotes Escherichia coli protein translocation by its association with the preprotei... more SecA ATPase promotes Escherichia coli protein translocation by its association with the preprotein or preprotein-SecB complex, anionic phospholipids, and the other core component of translocase, integral membrane protein SecYEG. Using ligand affinity blotting we demonstrate a direct interaction of SecA with SecY protein. Proteolysis and gene truncation or fusion studies were used to further define this interaction. Our results demonstrate that the carboxyl-terminal third of SecA protein binds to the amino-terminal 107 amino acid residues of SecY protein. The direct demonstration of these interactions culminate studies that have inferred an interaction between SecA and SecYEG, and they are consistent with studies suggesting that this region of SecA interacts with the inner membrane.

Research paper thumbnail of Nucleotide Binding Activity of SecA Homodimer Is Conformationally Regulated by Temperature and Altered by prlD and azi Mutations

Journal of Biological Chemistry, 2000

SecA ATPase is critical for protein translocation across the Escherichia coli inner membrane. To ... more SecA ATPase is critical for protein translocation across the Escherichia coli inner membrane. To understand this activity further, the high affinity nucleotide binding activity of SecA was characterized. We found that at 4°C SecA homodimer binds one ADP molecule with high affinity. This nucleotide binding activity was conformationally regulated by temperature: at low temperature SecA affinity for ADP was high with a slow exchange rate, whereas at high temperature the converse was true. Azi-and PrlD-SecA proteins that confer azide-resistant and signal sequence suppressor phenotypes were found to have reduced affinity for ADP and accelerated exchange rates compared with wild type SecA. Consistent with this observation, fluorescence and proteolysis studies indicated that these proteins had a conformationally relaxed state at a reduced temperature compared with SecA. The level of Azi-and PrlD-SecA protein was also elevated in inverted membrane vesicles where it displayed higher membrane ATPase activity. These results provide the first direct evidence for conformational regulation of the SecA-dependent nucleotide cycle, its alteration in azi and prlD mutants, and its relevance to in vivo protein export.

Research paper thumbnail of Revised Translation Start Site for secM Defines an Atypical Signal Peptide That Regulates Escherichia coli secA Expression

Journal of Bacteriology, 2000

The secretion-responsive regulation of Escherichia coli secA occurs by coupling its translation t... more The secretion-responsive regulation of Escherichia coli secA occurs by coupling its translation to the translation and secretion of an upstream regulator, secM (formerly geneX). We revise the translational start site for secM, defining a new signal peptide sequence with an extended amino-terminal region. Mutational studies indicate that certain atypical amino acyl residues within this extended region are critical for propersecA regulation.

Research paper thumbnail of Re-examination of the Role of the Amino-Terminus of SecA in Promoting Its Dimerization and Functional State

Biophysical Journal, 2009

The SecA nanomotor promotes protein translocation in eubacteria by binding both protein cargo and... more The SecA nanomotor promotes protein translocation in eubacteria by binding both protein cargo and the protein-conducting channel and by undergoing ATP-driven conformation cycles that drive this process. There are conflicting reports about whether SecA functions as a ...

Research paper thumbnail of Protein Secretion in Escherichia Coli

Annual Review of Microbiology, 1985

... 624 OLIVER The Basic Amino-Terminus Oligonucleotide-directed mutagenesis (149) has been used ... more ... 624 OLIVER The Basic Amino-Terminus Oligonucleotide-directed mutagenesis (149) has been used to remove one or both of the lysine residues in the signal sequence of the major outer membrane lipoprotein (Lpp) and replace them with neutral or acidic amino acids (76, 168 ...

Research paper thumbnail of Nucleotide Binding Activity of SecA Homodimer Is Conformationally Regulated by Temperature and Altered by prlD and azi Mutations

Journal of Biological Chemistry, 2000

SecA ATPase is critical for protein translocation across the Escherichia coli inner membrane. To ... more SecA ATPase is critical for protein translocation across the Escherichia coli inner membrane. To understand this activity further, the high affinity nucleotide binding activity of SecA was characterized. We found that at 4°C SecA homodimer binds one ADP molecule with high affinity. This nucleotide binding activity was conformationally regulated by temperature: at low temperature SecA affinity for ADP was high with a slow exchange rate, whereas at high temperature the converse was true. Azi-and PrlD-SecA proteins that confer azide-resistant and signal sequence suppressor phenotypes were found to have reduced affinity for ADP and accelerated exchange rates compared with wild type SecA. Consistent with this observation, fluorescence and proteolysis studies indicated that these proteins had a conformationally relaxed state at a reduced temperature compared with SecA. The level of Azi-and PrlD-SecA protein was also elevated in inverted membrane vesicles where it displayed higher membrane ATPase activity. These results provide the first direct evidence for conformational regulation of the SecA-dependent nucleotide cycle, its alteration in azi and prlD mutants, and its relevance to in vivo protein export.

Research paper thumbnail of The Variable Subdomain of Escherichia coli SecA Functions To Regulate SecA ATPase Activity and ADP Release

Journal of Bacteriology, 2012

Bacterial SecA proteins can be categorized by the presence or absence of a variable subdomain (VA... more Bacterial SecA proteins can be categorized by the presence or absence of a variable subdomain (VAR) located within nucleotidebinding domain II of the SecA DEAD motor. Here we show that VAR is dispensable for SecA function, since the VAR deletion mutant secA⌬519-547 displayed a wild-type rate of cellular growth and protein export. Loss or gain of VAR is extremely rare in the history of bacterial evolution, indicating that it appears to contribute to secA function within the relevant species in their natural environments. VAR removal also results in additional secA phenotypes: azide resistance (Azi r ) and suppression of signal sequence defects (PrlD). The SecA⌬(519 -547) protein was found to be modestly hyperactive for SecA ATPase activities and displayed an accelerated rate of ADP release, consistent with the biochemical basis of azide resistance. Based on our findings, we discuss models whereby VAR allosterically regulates SecA DEAD motor function at SecYEG.