Introduction of basic amino acid residues after the signal peptide inhibits protein translocation across the cytoplasmic membrane of Escherichia coli. Relation to the orientation of membrane proteins.

ABSTRACT In Escherichia coli, the SecB/SecA branch of the Sec pathway and the twin-arginine translocation (Tat) pathway represent two alternative possibilities for posttranslational translocation of proteins across the cytoplasmic membrane. Maintenance of pathway specificity was analyzed using a model precursor consisting of the mature part of the SecB-dependent maltose-binding protein (MalE) fused to the signal peptide of the Tat-dependent TorA protein. The TorA signal peptide selectively and specifically directed MalE into the Tat pathway. The characterization of a spontaneous TorA signal peptide mutant (TorA*), in which the two arginine residues in the c-region had been replaced by one leucine residue, showed that the TorA*-MalE mutant precursor had acquired the ability for efficiently using the SecB/SecA pathway. Despite the lack of the “Sec avoidance signal,” the mutant precursor was still capable of using the Tat pathway, provided that the kinetically favored Sec pathway was blocked. These results show that the h-region of the TorA signal peptide is, in principle, sufficiently hydrophobic for Sec-dependent protein translocation, and therefore, the positively charged amino acid residues in the c-region represent a major determinant for Tat pathway specificity. Tat-dependent export of TorA-MalE was significantly slower in the presence of SecB than in its absence, showing that SecB can bind to this precursor despite the presence of the Sec avoidance signal in the c-region of the TorA signal peptide, strongly suggesting that the function of the Sec avoidance signal is not the prevention of SecB binding; rather, it must be exerted at a later step in the Sec pathway.

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The Topology of the l-Arginine Exporter ArgO Conforms to an Nin-CoutConfiguration in Escherichia coli: Requirement for the Cytoplasmic N-Terminal Domain, Functional Helical Interactions, and an Aspartate Pair for ArgO Function

Journal of Bacteriology ◽

10.1128/jb.00423-16 ◽

2016 ◽

Vol 198 (23) ◽

pp. 3186-3199 ◽

Cited By ~ 4

Author(s):

Amit Pathania ◽

Arvind Kumar Gupta ◽

Swati Dubey ◽

Balasubramanian Gopal ◽

Abhijit A. Sardesai

Keyword(s):

Escherichia Coli ◽

Amino Acid ◽

Cytoplasmic Membrane ◽

Basic Amino Acid ◽

Intramolecular Interactions ◽

Membrane Topology ◽

Content Type ◽

E Coli ◽

Terminal Domain

ABSTRACTArgO and LysE are members of the LysE family of exporter proteins and ordinarily mediate the export ofl-arginine (Arg) inEscherichia coliandl-lysine (Lys) and Arg inCorynebacterium glutamicum, respectively. Under certain conditions, ArgO also mediates Lys export. To delineate the arrangement of ArgO in the cytoplasmic membrane ofE. coli, we have employed a combination of cysteine accessibilityin situ, alkaline phosphatase fusion reporters, and protein modeling to arrive at a topological model of ArgO. Our studies indicate that ArgO assumes an Nin-Coutconfiguration, potentially forming a five-transmembrane helix bundle flanked by a cytoplasmic N-terminal domain (NTD) comprising roughly its first 38 to 43 amino acyl residues and a short periplasmic C-terminal region (CTR). Mutagenesis studies indicate that the CTR, but not the NTD, is dispensable for ArgO functionin vivoand that a pair of conserved aspartate residues, located near the opposing edges of the cytoplasmic membrane, may play a pivotal role in facilitating transmembrane Arg flux. Additional studies on amino acid substitutions that impair ArgO functionin vivoand their derivatives bearing compensatory amino acid alterations indicate a role for intramolecular interactions in the Arg export mechanism, and some interactions are corroborated by normal-mode analyses. Lastly, our studies suggest that ArgO may exist as a monomerin vivo, thus highlighting the requirement for intramolecular interactions in ArgO, as opposed to interactions across multiple ArgO monomers, in the formation of an Arg-translocating conduit.IMPORTANCEThe orthologous proteins LysE ofC. glutamicumand ArgO ofE. colifunction as exporters of the basic amino acidsl-arginine andl-lysine and the basic amino acidl-arginine, respectively, and LysE can functionally substitute for ArgO when expressed inE. coli. Notwithstanding this functional equivalence, studies reported here show that ArgO possesses a membrane topology that is distinct from that reported for LysE, with substantial variation in the topological arrangement of the proximal one-third portions of the two exporters. Additional genetic andin silicostudies reveal the importance of (i) the cytoplasmic N-terminal domain, (ii) a pair of conserved aspartate residues, and (iii) potential intramolecular interactions in ArgO function and indicate that an Arg-translocating conduit is formed by a monomer of ArgO.

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Membrane Protein Degradation by FtsH Can Be Initiated from Either End

Journal of Bacteriology ◽

10.1128/jb.184.17.4775-4782.2002 ◽

2002 ◽

Vol 184 (17) ◽

pp. 4775-4782 ◽

Cited By ~ 52

Author(s):

Shinobu Chiba ◽

Yoshinori Akiyama ◽

Koreaki Ito

Keyword(s):

Escherichia Coli ◽

Amino Acid ◽

Membrane Proteins ◽

Membrane Protein ◽

Exact Sequence ◽

Integral Membrane Proteins ◽

Amino Acid Residues ◽

Aaa Atpase ◽

Membrane Bound ◽

Periplasmic Domain

ABSTRACT FtsH, a membrane-bound metalloprotease, with cytoplasmic metalloprotease and AAA ATPase domains, degrades both soluble and integral membrane proteins in Escherichia coli. In this paper we investigated how membrane-embedded substrates are recognized by this enzyme. We showed previously that FtsH can initiate processive proteolysis at an N-terminal cytosolic tail of a membrane protein, by recognizing its length (more than 20 amino acid residues) but not exact sequence. Subsequent proteolysis should involve dislocation of the substrates into the cytosol. We now show that this enzyme can also initiate proteolysis at a C-terminal cytosolic tail and that the initiation efficiency depends on the length of the tail. This mode of degradation also appeared to be processive, which can be aborted by a tightly folded periplasmic domain. These results indicate that FtsH can exhibit processivity against membrane-embedded substrates in either the N-to-C or C-to-N direction. Our results also suggest that some membrane proteins receive bidirectional degradation simultaneously. These results raise intriguing questions about the molecular directionality of the dislocation and proteolysis catalyzed by FtsH.

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Effect of alterations of basic amino acid residues of Escherichia coli heat-stable enterotoxin II on enterotoxicity.

Infection and Immunity ◽

10.1128/iai.62.6.2295-2301.1994 ◽

1994 ◽

Vol 62 (6) ◽

pp. 2295-2301 ◽

Cited By ~ 12

Author(s):

Y Fujii ◽

Y Okamuro ◽

S Hitotsubashi ◽

A Saito ◽

N Akashi ◽

...

Keyword(s):

Escherichia Coli ◽

Amino Acid ◽

Basic Amino Acid ◽

Amino Acid Residues ◽

Heat Stable

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A Truncated Bacillus subtilis SecA Protein Consisting of the N-Terminal 234 Amino Acid Residues Forms a Complex with Escherichia coli SecA51(ts) Protein and Complements the Protein Translocation Defect of the secA51 Mutant1

The Journal of Biochemistry ◽

10.1093/oxfordjournals.jbchem.a124677 ◽

1994 ◽

Vol 116 (6) ◽

pp. 1287-1294 ◽

Cited By ~ 8

Author(s):

Hiromu Takamatsu ◽

Akitaka Nakane ◽

Akihiro Oguro ◽

Yoshito Sadaie ◽

Kouji Nakamura ◽

...

Keyword(s):

Escherichia Coli ◽

Bacillus Subtilis ◽

Amino Acid ◽

Protein Translocation ◽

Amino Acid Residues

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Computational redesign of the lipid-facing surface of the outer membrane protein OmpA

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1501836112 ◽

2015 ◽

Vol 112 (31) ◽

pp. 9632-9637 ◽

Cited By ~ 15

Author(s):

James A. Stapleton ◽

Timothy A. Whitehead ◽

Vikas Nanda

Keyword(s):

Escherichia Coli ◽

Amino Acid ◽

Membrane Proteins ◽

Cell Membrane ◽

Membrane Protein ◽

Computational Design ◽

Residue Level ◽

Membrane Insertion ◽

Amino Acid Residues ◽

Design Rules

Advances in computational design methods have made possible extensive engineering of soluble proteins, but designed β-barrel membrane proteins await improvements in our understanding of the sequence determinants of folding and stability. A subset of the amino acid residues of membrane proteins interact with the cell membrane, and the design rules that govern this lipid-facing surface are poorly understood. We applied a residue-level depth potential for β-barrel membrane proteins to the complete redesign of the lipid-facing surface ofEscherichia coliOmpA. Initial designs failed to fold correctly, but reversion of a small number of mutations indicated by backcross experiments yielded designs with substitutions to up to 60% of the surface that did support folding and membrane insertion.

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Primary- and secondary-structural analysis of a unique prokaryotic metallothionein from a Synechococcus sp. cyanobacterium

Biochemical Journal ◽

10.1042/bj2510691 ◽

1988 ◽

Vol 251 (3) ◽

pp. 691-699 ◽

Cited By ~ 106

Author(s):

R W Olafson ◽

W D McCubbin ◽

C M Kay

Keyword(s):

Amino Acid ◽

Metal Binding ◽

Helical Structure ◽

Basic Amino Acid ◽

Cysteine Residues ◽

Amino Acid Residues ◽

C Terminus ◽

Empirical Relations ◽

Heavy Metal Binding ◽

Synechococcus Sp

Biochemical and physiological studies of Synechococcus cyanobacteria have indicated the presence of a low-Mr heavy-metal-binding protein with marked similarity to eukaryotic metallothioneins (MTs). We report here the characterization of a Synechococcus prokaryotic MT isolated by gel-permeation and reverse-phase chromatography. The large number of variants of this molecule found during chromatographic separation could not be attributed to the presence of major isoproteins as assessed by amino acid analysis and amino acid sequencing of isoforms. Two of the latter were shown to have identical primary structures that differed substantially from the well-described eukaryotic MTs. In addition to six long-chain aliphatic residues, two aromatic residues were found adjacent to one another near the centre of the molecule, making this the most hydrophobic MT to be described. Other unusual features included a pair of histidine residues located in repeating Gly-His-Thr-Gly sequences near the C-terminus and a complete lack of association of hydroxylated residues with cysteine residues, as is commonly found in eukaryotes. Similarly, aside from a single lysine residue, no basic amino acid residues were found adjacent to cysteine residues in the sequence. Most importantly, sequence alignment analyses with mammalian, invertebrate and fungal MT sequences showed no statistically significant homology aside from the presence of Cys-Xaa-Cys structures common to all MTs. On the other hand, like other MTs, the prokaryotic molecule appears to be free of alpha-helical structure but has a considerable amount of beta-structure, as predicted by both c.d. measurements and the Chou & Fasman empirical relations. Considered together, these data suggested that some similarity between the metal-thiolate clusters of the prokaryote and eukaryote MTs may exist.

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