Effects of altered TatC proteins on protein secretion efficiency via the twin-arginine translocation pathway of Bacillus subtilis

Protein translocation via the Tat machinery in thylakoids and bacteria occurs through a cooperation between the TatA, TatB and TatC subunits, of which the TatC protein forms the initial Tat substrate-binding site. The Bacillus subtilis Tat machinery lacks TatB and comprises two separate TatAC complexes with distinct substrate specificities: PhoD is secreted by the TatAdCd complex, whereas YwbN is secreted by the TatAyCy complex. To study the role of the Gram-positive TatC proteins in Tat-dependent protein secretion efficiency, we applied several genetic engineering approaches to modify and analyse the B. subtilis TatCd and TatCy proteins. Cytoplasmic and transmembrane domain exchange between TatCd and TatCy resulted in stable chimeric proteins that were unable to secrete both known substrates of the B. subtilis Tat system. Site-directed mutagenesis of conserved residues in the N-terminal part of both TatC proteins revealed significant differences in the degree of importance of these residues between TatCd, TatCy and Escherichia coli TatC. In addition, two small C-terminal deletions in TatCy completely abolished YwbN translocation, indicating that this terminus is essential for Tat translocation activity. Important differences from previous observations for E. coli TatC and implications for substrate binding and translocation are discussed.

Role of Individual Positive Charges in the Membrane Orientation and Activity of Transporters of the Small Multidrug Resistance Family

The effect of individual positively charged residues on the orientation in the membrane was analyzed in three dual-topology transporters of the small. multidrug resistance (SMR) family: AAVE4701aave of Acidovorax avenae, EMREecol of Escherichia coli, and RRUA0272rrub of Rhodospirillum rubrum. It is shown that (i) individual positive charges have different impacts on the orientation, (ii) positive charges that are conserved in the three different proteins do not have the same impact on the orientation, (iii) positive charges in odd- and even-numbered loops have different impacts, (iv) for some, but not all, the impact depends on the presence of other positive charges, and (v) proteins from which all positive charges are removed in some cases are dual-topology proteins and in other cases have a single orientation. A small number of positive charges placed in the loops of the latter proteins results in the violation of the so-called positive-inside rule that has been reported previously [Kolbusz, M. A., et al. (2010) J. Mol. Biol. 402, 127-138]. We conclude that each positive charge shifts the distribution between the two orientations toward the state that has the positive charge in the cytoplasm but that intrinsic factors other than positive charges determine the orientation as well. The ability of the mutants of AAVE4701aave and EMREecol to confer resistance against ethidium bromide revealed an essential role in catalysis for a conserved pair of positive charges in the second loop. No significant relation between activity and the relative orientation of the monomeric subunits in the dimer could be demonstrated

Orientation of Small Multidrug Resistance Transporter Subunits in the Membrane:Correlation with the Positive-Inside Rule

Small multidrug resistance (SMR) transport proteins provide a model for the evolution of larger two-domain transport proteins. The orientation in the membrane of 27 proteins from the SMR family was determined using the reporter fusion technique. Nine members were encoded monocistronically (singles) and shown to insert in both orientations (dual topology). Eighteen members were encoded in pairs on the chromosome and shown to insert in fixed orientations; the two proteins in each pair invariably had opposite orientations in the membrane. Interaction between the two proteins in pairs was demonstrated by copurification. The orientation in the membrane of either protein in the pair was affected only marginally by the presence of the other protein. For the proteins in pairs, the orientation in the membrane correlated well with the distribution of positively charges residues (R + K) over the cytoplasmic and extracellular loops (positive-inside rule). In contrast, dual-topology insertion of the singles was predicted less well by the positive-inside rule. Three singles were predicted to insert in a single orientation with the N-terminus and the C-terminus at the extracellular side of the membrane. Analysis of charge distributions suggests the requirement of a threshold number of charges in the cytoplasmic loops for the positive-inside rule to be of predictive value. It is concluded that a combined analysis of gene organization on the chromosome and phylogeny is sufficient to distinguish between fixed or dual topology of SMR members and, probably, similar types of membrane proteins. The positive-inside rule can be used to predict the orientation of members in pairs, but is not suitable as a sole predictor of dual topology. (C) 2010 Elsevier Ltd. All rights reserved