Sharon Mendel

The N-terminal domain of the regulatory subunit is sufficient for complete activation of acetohydroxyacid synthase III from Escherichia coli

We have previously proposed a model for the fold of the N-terminal domain of the small, regulatory subunit (SSU) of acetohydroxyacid synthase isozyme III. The fold is an alpha-beta sandwich with betaalphabetabetaalphabeta topology, structurally homologous to the C-terminal regulatory domain of 3-phosphoglycerate dehydrogenase. We suggested that the N-terminal domains of a pair of SSUs interact in the holoenzyme to form two binding sites for the feedback inhibitor valine in the interface between them. The model was supported by mutational analysis and other evidence. We have now examined the role of the C-terminal portion of the SSU by construction of truncated polypeptides (lacking 35, 48, 80, 95, or 112 amino acid residues from the C terminus) and examining the properties of holoenzymes reconstituted using these constructs. The Delta35, Delta48, and Delta80 constructs all lead to essentially complete activation of the catalytic subunits. The Delta80 construct, corresponding to the putative N-terminal domain, has the highest level of affinity for the catalytic subunits and leads to a reconstituted enzyme with k(cat)/K(M) about twice that of the wild-type enzyme. On the other hand, none of these constructs binds valine or leads to a valine-sensitive enzyme on reconstitution. The enzyme reconstituted with the Delta80 construct does not bind valine, either. The N-terminal portion (about 80 amino acid residues) of the SSU is thus necessary and sufficient for recognition and activation of the catalytic subunits, but the C-terminal half of the SSU is required for valine binding and response. We suggest that the C-terminal region of the SSU contributes to monomer-monomer interactions, and provide additional experimental evidence for this suggestion.

The twin-arginine translocation (Tat) systems from Bacillus subtilis display a conserved mode of complex organization and similar substrate recognition requirements.

The twin arginine translocation (Tat) system transports folded proteins across the bacterial plasma membrane. In Gram‐negative bacteria, membrane‐bound TatABC subunits are all essential for activity, whereas Gram‐positive bacteria usually contain only TatAC subunits. In Bacillus subtilis, two TatAC‐type systems, TatAdCd and TatAyCy, operate in parallel with different substrate specificities. Here, we show that they recognize similar signal peptide determinants. Both systems translocate green fluorescent protein fused to three distinct Escherichia coli Tat signal peptides, namely DmsA, AmiA and MdoD, and mutagenesis of the DmsA signal peptide confirmed that both Tat pathways recognize similar targeting determinants within Tat signals. Although another E. coli Tat substrate, trimethylamine N‐oxide reductase, was translocated by TatAdCd but not by TatAyCy, we conclude that these systems are not predisposed to recognize only specific Tat signal peptides, as suggested by their narrow substrate specificities in B. subtilis. We also analysed complexes involved in the second Tat pathway in B. subtilis, TatAyCy. This revealed a discrete TatAyCy complex together with a separate, homogeneous, ∼ 200 kDa TatAy complex. The latter complex differs significantly from the corresponding E. coli TatA complexes, pointing to major structural differences between Tat complexes from Gram‐negative and Gram‐positive organisms. Like TatAd, TatAy is also detectable in the form of massive cytosolic complexes.

Expression of the bifunctional Bacillus subtilis TatAd protein in Escherichia coli reveals distinct TatA/B-family and TatB-specific domains

In the Tat protein export pathway of Gram-negative bacteria, TatA and TatB are homologous proteins that carry out distinct and essential functions in separate sub-complexes. In contrast, Gram-positive Tat systems usually lack TatB and the TatA protein is bifunctional. We have used a mutagenesis approach to delineate TatA/B-type domains in the bifunctional TatAd protein from Bacillus subtilis. This involved expression of mutated TatAd variants in Escherichia coli and tests to determine whether the variants could function as TatA or TatB by complementing E. colitatA and/or tatB mutants. We show that mutations in the C-terminal half of the transmembrane span and the subsequent FGP ‘hinge’ motif are critical for TatAd function with its partner TatCd subunit, and the same determinants are required for complementation of either tatA or tatB mutants in Escherichia coli. This is thus a critical domain in both TatA and TatB proteins. In contrast, substitution of a series of residues at the N-terminus specifically blocks the ability of TatAd to substitute for E. coli TatB. The results point to the presence of a universally conserved domain in the TatA/B-family, together with a separate N-terminal domain that is linked to the TatB-type function in Gram-negative bacteria.

Targeting of Proteins by the Twin-Arginine Translocation System in Bacteria and Chloroplasts

Publisher Summary Translocation of proteins is an important process in essentially all living organisms and there are two main pathways for the export of proteins in most free-living bacteria: the secretory (Sec) pathway and the twin-Arg translocation (Tat) pathway. This chapter focuses on the twin-Arg translocation (Tat) pathway with the aim of describing the structure, function, and mechanism of this unusual system. The two pathways differ in fundamental respects, particularly in the folding state of the substrate; Sec substrates are delivered into the Sec pathway in an unfolded state and are maintained in this state for the duration of the targeting pathway, while the Tat system is highly unusual in transporting its substrates in a folded state. The Tat translocase is present in many, but not all bacteria, and is widely distributed in archaea; however, is not present in animals or yeasts. Many prokaryotes use the Tat pathway predominantly for the secretion of redox proteins, but an analysis of the predicted substrates suggests that certain bacteria and archaea secrete mainly nonredox proteins via the Tat system, suggesting that the Tat system is predominantly used for the export of two types of protein: those that are obliged to fold prior to export and those that cannot be transported by the Sec pathway for other reasons. Most lumenal proteins in plants are transported by the Tat pathway in chloroplasts, indicating a critical role for this system in chloroplast biogenesis. It is widely accepted, though perhaps not formally proven, that the Tat system transports large proteins in a folded form, but the actual translocation mechanism is still poorly understood.