Functional Analysis of Semi-conserved Transit Peptide Motifs and Mechanistic Implications in Precursor Targeting and Recognition.

Over 95% of plastid proteins are nuclear-encoded as their precursors containing an N-terminal extension known as the transit peptide (TP). Although highly variable, TPs direct the precursors through a conserved, posttranslational mechanism involving translocons in the outer (TOC) and inner envelope (TOC). The organelle import specificity is mediated by one or more components of the Toc complex. However, the high TP diversity creates a paradox on how the sequences can be specifically recognized. An emerging model of TP design is that they contain multiple loosely conserved motifs that are recognized at different steps in the targeting and transport process. Bioinformatics has demonstrated that many TPs contain semi-conserved physicochemical motifs, termed FGLK. In order to characterize FGLK motifs in TP recognition and import, we have analyzed two well-studied TPs from the precursor of RuBisCO small subunit (SStp) and ferredoxin (Fdtp). Both SStp and Fdtp contain two FGLK motifs. Analysis of large set mutations (∼85) in these two motifs using in vitro, in organello, and in vivo approaches support a model in which the FGLK domains mediate interaction with TOC34 and possibly other TOC components. In vivo import analysis suggests that multiple FGLK motifs are functionally redundant. Furthermore, we discuss how FGLK motifs are required for efficient precursor protein import and how these elements may permit a convergent function of this highly variable class of targeting sequences.

Myristoylome profiling reveals a concerted mechanism of ARF GTPase deacylation by the bacterial protease IpaJ.

N-myristoylation is an essential fatty acid modification that governs the localization and activity of cell signaling enzymes, architectural proteins, and immune regulatory factors. Despite its importance in health and disease, there are currently no methods for reversing protein myristoylation in vivo. Recently, the Shigella flexneri protease IpaJ was found to cleave myristoylated glycine of eukaryotic proteins, yet the discriminatory mechanisms of substrate selection required for targeted demyristoylation have not yet been evaluated. Here, we performed global myristoylome profiling of cells treated with IpaJ under distinct physiological conditions. The protease is highly promiscuous among diverse N-myristoylated proteins in vitro but is remarkably specific to Golgi-associated ARF/ARL family GTPases during Shigella infection. Reconstitution studies revealed a mechanistic framework for substrate discrimination based on IpaJ's function as a GTPase "effector" of bacterial origin. We now propose a concerted model for IpaJ function that highlights its potential for programmable demyristoylation in vivo.

Bacteria fighting back: how pathogens target and subvert the host innate immune system.

The innate immune system has evolved under selective pressure since the radiation of multicellular life approximately 600 million years ago. Because of this long history, innate immune mechanisms found in modern eukaryotic organisms today are highly complex but yet built from common molecular strategies. It is now clear that evolution has selected a conserved set of antimicrobial peptides as well as pattern-recognition receptors (PRRs) that initiate cellular-based signals as a first line of defense against invading pathogens. Conversely, microbial pathogens employ their own strategies in order to evade, inhibit, or otherwise manipulate the innate immune response. Here, we discuss recent discoveries that have changed our view of immune modulatory mechanisms employed by bacterial pathogens, focusing specifically on the initial sites of microbial recognition and extending to host cellular signal transduction, proinflammatory cytokine production, and alteration of protein trafficking and secretion.

Differential transit peptide recognition during preprotein binding and translocation into flowering plant plastids.

Despite the availability of thousands of transit peptide (TP) primary sequences, the structural and/or physicochemical properties that determine TP recognition by components of the chloroplast translocon are not well understood. By combining a series of in vitro and in vivo experiments, we reveal that TP recognition is determined by sequence-independent interactions and vectorial-specific recognition domains. Using both native and reversed TPs for two well-studied precursors, small subunit of ribulose-1,5-bis-phosphate carboxylase/oxygenase, and ferredoxin, we exposed these two modes of recognition. Toc34 receptor (34-kD subunit of the translocon of the outer envelope) recognition in vitro, preprotein binding in organellar, precursor binding in vivo, and the recognition of TPs by the major stromal molecular motor Hsp70 are specific for the physicochemical properties of the TP. However, translocation in organellar and in vivo demonstrates strong specificity to recognition domain organization. This organization specificity correlates with the N-terminal placement of a strong Hsp70 recognition element. These results are discussed in light of how individual translocon components sequentially interact with the precursor during binding and translocation and helps explain the apparent lack of sequence conservation in chloroplast TPs.

Correlative light and electron microscopy (CLEM) as a tool to visualize microinjected molecules and their eukaryotic sub-cellular targets.

The eukaryotic cell relies on complex, highly regulated, and functionally distinct membrane bound compartments that preserve a biochemical polarity necessary for proper cellular function. Understanding how the enzymes, proteins, and cytoskeletal components govern and maintain this biochemical segregation is therefore of paramount importance. The use of fluorescently tagged molecules to localize to and/or perturb subcellular compartments has yielded a wealth of knowledge and advanced our understanding of cellular regulation. Imaging techniques such as fluorescent and confocal microscopy make ascertaining the position of a fluorescently tagged small molecule relatively straightforward, however the resolution of very small structures is limited. On the other hand, electron microscopy has revealed details of subcellular morphology at very high resolution, but its static nature makes it difficult to measure highly dynamic processes with precision. Thus, the combination of light microscopy with electron microscopy of the same sample, termed Correlative Light and Electron Microscopy (CLEM), affords the dual advantages of ultrafast fluorescent imaging with the high-resolution of electron microscopy. This powerful technique has been implemented to study many aspects of cell biology. Since its inception, this procedure has increased our ability to distinguish subcellular architectures and morphologies at high resolution. Here, we present a streamlined method for performing rapid microinjection followed by CLEM (Fig. 1). The microinjection CLEM procedure can be used to introduce specific quantities of small molecules and/or proteins directly into the eukaryotic cell cytoplasm and study the effects from millimeter to multi-nanometer resolution (Fig. 2). The technique is based on microinjecting cells grown on laser etched glass gridded coverslips affixed to the bottom of live cell dishes and imaging with both confocal fluorescent and electron microscopy. Localization of the cell(s) of interest is facilitated by the grid pattern, which is easily transferred, along with the cells of interest, to the Epon resin used for immobilization of samples and sectioning prior to electron microscopy analysis (Fig. 3). Overlay of fluorescent and EM images allows the user to determine the subcellular localization as well as any morphological and/or ultrastructural changes induced by the microinjected molecule of interest (Fig. 4). This technique is amenable to time points ranging from ≤5 s up to several hours, depending on the nature of the microinjected sample.

Express your LOV: an engineered flavoprotein as a reporter for protein expression and purification.

In this work, we describe the utility of Light, Oxygen, or Voltage-sensing (LOV) flavoprotein domains from plant phototropins as a reporter for protein expression and function. Specifically, we used iLOV, an enhanced and more photostable variant of LOV. A pET-based plasmid for protein expression was constructed, encoding a C terminal iLOV-octahistidine (His8)-tag and a HRV 3C protease cleavage recognition site. Ten different proteins, with various sub-cellular locations, were cloned into the plasmid, creating iLOV-His8 tag fusions. To test protein expression and how iLOV could be used as a reporter, the proteins were expressed in three different cell lines, in four different culture media, at two different temperatures. To establish whether the presence of the iLOV tag could have an impact on the functionality, one of the proteins, EspG, was over-expressed and purified. EspG is an "effector" protein normally produced by enterohemorrhagic E. coli strains and "injected" into host cells via the T3SS. We tested functionality of EspG-iLOV fusion by performing functional studies of EspG in mammalian host cells. When EspG-iLOV was microinjected into the host cell, the Golgi apparatus was completely disrupted as had previously been observed for EspG.

The assembly of a GTPase-kinase signalling complex by a bacterial catalytic scaffold.

The fidelity and specificity of information flow within a cell is controlled by scaffolding proteins that assemble and link enzymes into signalling circuits. These circuits can be inhibited by bacterial effector proteins that post-translationally modify individual pathway components. However, there is emerging evidence that pathogens directly organize higher-order signalling networks through enzyme scaffolding, and the identity of the effectors and their mechanisms of action are poorly understood. Here we identify the enterohaemorrhagic Escherichia coli O157:H7 type III effector EspG as a regulator of endomembrane trafficking using a functional screen, and report ADP-ribosylation factor (ARF) GTPases and p21-activated kinases (PAKs) as its relevant host substrates. The 2.5 Å crystal structure of EspG in complex with ARF6 shows how EspG blocks GTPase-activating-protein-assisted GTP hydrolysis, revealing a potent mechanism of GTPase signalling inhibition at organelle membranes. In addition, the 2.8 Å crystal structure of EspG in complex with the autoinhibitory Iα3-helix of PAK2 defines a previously unknown catalytic site in EspG and provides an allosteric mechanism of kinase activation by a bacterial effector. Unexpectedly, ARF and PAKs are organized on adjacent surfaces of EspG, indicating its role as a 'catalytic scaffold' that effectively reprograms cellular events through the functional assembly of GTPase-kinase signalling complex.

Nano-scale characterization of the dynamics of the chloroplast Toc translocon.

Translocons are macromolecular nano-scale machines that facilitate the selective translocation of proteins across membranes. Although common in function, different translocons have evolved diverse molecular mechanisms for protein translocation. Subcellular organelles of endosymbiotic origin such as the chloroplast and mitochondria had to evolve/acquire translocons capable of importing proteins whose genes were transferred to the host genome. These gene products are expressed on cytosolic ribosomes as precursor proteins and targeted back to the organelle by an N-terminal extension called the transit peptide or presequence. In chloroplasts the transit peptide is specifically recognized by the Translocon of the Outer Chloroplast membrane (Toc) which is composed of receptor GTPases that potentially function as gate-like switches, where GTP binding and hydrolysis somehow facilitate preprotein binding and translocation. Compared to other translocons, the dynamics of the Toc translocon are probably more complex and certainly less understood. We have developed biochemical/biophysical, imaging, and computational techniques to probe the dynamics of the Toc translocon at the nanoscale. In this chapter we provide detailed protocols for kinetic and binding analysis of precursor interactions in organeller, measurement of the activity and nucleotide binding of the Toc GTPases, native electrophoretic analysis of the assembly/organization of the Toc complex, visualization of the distribution and mobility of Toc apparatus on the surface of chloroplasts, and conclude with the identification and molecular modeling Toc75 POTRA domains. With these new methodologies we discuss future directions of the field.

In vitro comparative kinetic analysis of the chloroplast Toc GTPases.

A unique aspect of protein transport into plastids is the coordinate involvement of two GTPases in the translocon of the outer chloroplast membrane (Toc). There are two subfamilies in Arabidopsis, the small GTPases (Toc33 and Toc34) and the large acidic GTPases (Toc90, Toc120, Toc132, and Toc159). In chloroplasts, Toc34 and Toc159 are implicated in precursor binding, yet mechanistic details are poorly understood. How the GTPase cycle is modulated by precursor binding is complex and in need of careful dissection. To this end, we have developed novel in vitro assays to quantitate nucleotide binding and hydrolysis of the Toc GTPases. Here we present the first systematic kinetic characterization of four Toc GTPases (cytosolic domains of atToc33, atToc34, psToc34, and the GTPase domain of atToc159) to permit their direct comparison. We report the KM, Vmax, and Ea values for GTP hydrolysis and the Kd value for nucleotide binding for each protein. We demonstrate that GTP hydrolysis by psToc34 is stimulated by chloroplast transit peptides; however, this activity is not stimulated by homodimerization and is abolished by the R133A mutation. Furthermore, we show peptide stimulation of hydrolytic rates are not because of accelerated nucleotide exchange, indicating that transit peptides function as GTPase-activating proteins and not guanine nucleotide exchange factors in modulating the activity of psToc34. Finally, by using the psToc34 structure, we have developed molecular models for atToc33, atToc34, and atToc159G. By combining these models with the measured enzymatic properties of the Toc GTPases, we provide new insights of how the chloroplast protein import cycle may be regulated.