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The type VI secretion system (T6SS) with diversified functions is widely distributed in pathogenic Proteobacteria. The IcmF (intracellular multiplication protein F) family protein TssM is a conserved T6SS inner membrane protein. Despite the conservation of its Walker A nucleotide-binding motif, the NTPase activity of TssM and its role in T6SS remain obscure. In this study, we characterized TssM in the plant pathogen Agrobacterium tumefaciens and provided the first biochemical evidence for TssM exhibiting ATPase activity to power the secretion of the T6SS hallmark protein, hemolysin-coregulated protein (Hcp). Amino acid substitutions in the Walker A motif of TssM caused reduced ATP binding and hydrolysis activity. Importantly, we discovered the Walker B motif of TssM and demonstrated that it is critical for ATP hydrolysis activity. Protein-protein interaction studies and protease susceptibility assays indicated that TssM undergoes an ATP binding-induced conformational change and that subsequent ATP hydrolysis is crucial for recruiting Hcp to interact with the periplasmic domain of the TssM-interacting protein TssL (an IcmH/DotU family protein) into a ternary complex and mediating Hcp secretion. Our findings strongly argue that TssM functions as a T6SS energizer to recruit Hcp into the TssM-TssL inner membrane complex prior to Hcp secretion across the outer membrane.

The β(2) subunit of class Ia ribonucleotide reductase (RNR) contains a diferric tyrosyl radical cofactor (Fe(2)(III)-Tyr(•)) that is essential for nucleotide reduction. The β(2) subunit of Saccharomyces cerevisiae is a heterodimer of Rnr2 (β) and Rnr4 (β'). Although only β is capable of iron binding and Tyr(•) formation, cells lacking β' are either dead or exhibit extremely low Tyr(•) levels and RNR activity depending on genetic backgrounds. Here, we present evidence supporting the model that β' is required for iron loading and Tyr(•) formation in β in vivo via a pathway that is likely dependent on the cytosolic monothiol glutaredoxins Grx3/Grx4 and the Fe-S cluster protein Dre2. rnr4 mutants are defective in iron loading into nascent β and are hypersensitive to iron depletion and the Tyr(•)-reducing agent hydroxyurea. Transient induction of β' in a GalRNR4 strain leads to a concomitant increase in iron loading and Tyr(•) levels in β. Tyr(•) can also be rapidly generated using endogenous iron when permeabilized Δrnr4 spheroplasts are supplemented with recombinant β' and is inhibited by adding an iron chelator prior to, but not after, β' supplementation. The growth defects of rnr4 mutants are enhanced by deficiencies in grx3/grx4 and dre2. Moreover, depletion of Dre2 in GalDRE2 cells leads to a decrease in both Tyr(•) levels and ββ' activity. This result, in combination with previous findings that a low level of Grx3/4 impairs RNR function, strongly suggests that Grx3/4 and Dre2 serve in the assembly of the deferric Tyr(•) cofactor in RNR.

The Trypanosoma brucei genome has four highly similar genes encoding sphingolipid synthases (TbSLS1-4). TbSLSs are polytopic membrane proteins that are essential for viability of the pathogenic bloodstream stage of this human protozoan parasite and, consequently, can be considered as potential drug targets. TbSLS4 was shown previously to be a bifunctional sphingomyelin/ethanolamine phosphorylceramide synthase, whereas functions of the others were not characterized. Using a recently described liposome-supplemented cell-free synthesis system, which eliminates complications from background cellular activities, we now unambiguously define the enzymatic specificity of the entire gene family. TbSLS1 produces inositol phosphorylceramide, TbSLS2 produces ethanolamine phosphorylceramide, and TbSLS3 is bifunctional, like TbSLS4. These findings indicate that TbSLS1 is uniquely responsible for synthesis of inositol phosphorylceramide in insect stage parasites, in agreement with published expression array data (17). This approach also revealed that the Trypanosoma cruzi ortholog (TcSLS1) is a dedicated inositol phosphorylceramide synthase. The cell-free synthesis system allowed rapid optimization of the reaction conditions for these enzymes and site-specific mutagenesis to alter end product specificity. A single residue at position 252 (TbSLS1, Ser(252); TbSLS3, Phe(252)) strongly influences enzymatic specificity. We also have used this system to demonstrate that aureobasidin A, a potent inhibitor of fungal inositol phosphorylceramide synthases, does not significantly affect any of the TbSLS activities, consistent with the phylogenetic distance of these two clades of sphingolipid synthases. These results represent the first application of cell-free synthesis for the rapid preparation and functional annotation of integral membrane proteins and thus illustrate its utility in studying otherwise intractable enzyme systems.