Toc34 is a transmembrane protein located in the outer envelope membrane of chloroplasts and involved in transit peptide recognition. The cytosolic region of Toc34 reveals 34% alpha-helical and 26% beta-strand structure and is stabilized by intramolecular electrostatic interaction. Toc34 binds both chloroplast preproteins and isolated transit peptides in a guanosine triphosphate- (GTP-) dependent mechanism. In this study we demonstrate that the soluble, cytosolic domain of Toc34 (Toc34deltaTM) functions as receptor in vitro and is capable to compete with the import of the preprotein of the small subunit (preSSU) of ribulose-1,5-bisphosphate carboxylase-oxygenase into chloroplasts in a GTP-dependent manner. We have developed a biosensor assay to study the interaction of Toc34deltaTM with purified preproteins and transit peptides. The results are compared with the interactions of both a full-size preprotein and the transit peptide of preSSU with the translocon of the outer envelope of chloroplasts (Toc complex) in situ. Several mutants of the transit peptide of preSSU were evaluated to identify amino acid segments that are specifically recognized by Toc34. We present a model of how Toc34 may recognize the transit peptide and discuss how this interaction may facilitate interaction and translocation of preproteins via the Toc complex in vivo.

Both authors contributed equally to this work. SummaryWe have recently isolated and identi®ed a novel mitochondrial metalloprotease, pre-sequence protease (PreP) from potato and shown that it degrades mitochondrial pre-sequences. PreP belongs to the pitrilysin protease family and contains an inverted zinc-binding motif. To further investigate the degradation of targeting peptides, we have overexpressed the Arabidopsis thaliana homologue of PreP, zinc metalloprotease (Zn-MP), in Escherichia coli. We have characterized the recombinant Zn-MP with respect to its catalytic site, substrate speci®city and intracellular localization. Mutagenesis studies of the residues involved in metal binding identi®ed the histidines and the proximal glutamate as essential residues for the proteolytic activity. Substrate speci®city studies showed that the Zn-MP has the ability to degrade both mitochondrial pre-sequences and chloroplastic transit peptides, as well as other unstructured peptides. The Zn-MP does not recognize an amino acid sequence per se. Immunological studies and proteolytic activity measurements in isolated mitochondria and chloroplasts revealed the presence of the Zn-MP in both organelles. Furthermore, the Zn-MP was found to be dually imported to both mitochondria and chloroplasts in vitro. In summary, our data show that the Zn-MP is present and serves the same function in chloroplasts as in mitochondria ± degradation of targeting peptides.

We report an approach that extends the applicability of ultrasensitive force-gradient detection of magnetic resonance to samples with spin-lattice relaxation times (T 1 ) as short as a single cantilever period. To demonstrate the generality of the approach, which relies on detecting either cantilever frequency or phase, we used it to detect electron spin resonance from a T 1 = 1 ms nitroxide spin probe in a thin film at 4.2 K and 0.6 T. By using a custom-fabricated cantilever with a 4 μm-diameter nickel tip, we achieve a magnetic resonance sensitivity of 400 Bohr magnetons in a 1 Hz bandwidth. A theory is presented that quantitatively predicts both the lineshape and the magnitude of the observed cantilever frequency shift as a function of field and cantilever-sample separation. Good agreement was found between nitroxide T 1 's measured mechanically and inductively, indicating that the cantilever magnet is not an appreciable source of spin-lattice relaxation here. We suggest that the new approach has a number of advantages that make it well suited to push magnetic resonance detection and imaging of nitroxide spin labels in an individual macromolecule to single-spin sensitivity.MRFM | ESR | TEMPAMINE | mechanically detected magnetic resonance | molecular structure imaging A generally applicable approach for determining the tertiary structure of an individual macromolecule in vitro at angstrom or subangstrom resolution would create exciting opportunities for answering many longstanding questions in molecular biology. For macromolecules too large to characterize by NMR or X-ray diffraction, the tertiary structure of proteins (1-3), nucleic acids (4, 5), and biomolecular assemblies (6, 7) can be explored by using inductively-detected electron spin resonance (ESR) to measure distances between pairs of attached spin labels (2-5, 7, 8). These studies, however, require bulk quantities of sample (9) and demand multiple experiments with spin labels attached to different locations in the target macromolecule. Mechanical detection and imaging of single-electron spins has been demonstrated, in E centers in gamma-irradiated quartz (10), and it is natural to explore applying magnetic resonance force microscopy (MRFM) (11-15) to map the locations of individual spin labels attached to a single biomacromolecule.The ultimate limit of imaging resolution in MRFM is set by the intrinsic linewidth of the resonance and the applied magnetic field gradient. For a 0.1 mT homogeneous linewidth, typical of the organic radical studied here, a gradient of 4 × 10 6 T/m allows selective excitation of individual spin labels only 0.025 nm apart. A magnetic field gradient this large has recently been demonstrated in an MRFM experiment by using ferromagnetic pillars fabricated by electron-beam lithography (15). The force sensitivity required to detect single electrons in this gradient is 40 aN, above the minimum detectable force (in 1 Hz bandwidth) of 5 − 10 aN reported for a high-compliance cantilever operated with its metalized leading edge above ...

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 K M , V max , and E a values for GTP hydrolysis and the K d 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.Both mitochondria and chloroplasts arose through endosymbiotic events that followed phagocytotic internalization of either a free-living ␣-proteobacteria or cyanobacteria. During the course of evolution, in both cases, the vast majority of the endosymbiont genes was relocated to the host nucleus (1). Moreover, in higher plants, most of the proteome from both organelles is derived from nuclearly encoded proteins that are translated on free cytosolic ribosomes and then post-translationally translocated into the organelle via distinct protein complexes located in each of the two membranes that enclose these organelles (2, 3). These mitochondrial and chloroplast translocators are denoted as Tim/Tom and Tic/Toc, respectively (Translocator of the inner/outer membrane of mitochondria/chloroplast). The Toc 5 complex consists of three key proteins denoted by their apparent molecular masses, Toc34, Toc75, and Toc159, and most likely exist with a stoichiometry of 4:4:1, respectively (4). Proteins targeted from the cytosol to these mitochondrial and chloroplast translocators require additional "information" in the form of an N-terminal targeting sequence known as a presequence and transit peptide, respectively (1). This additional targeting sequence has been added during evolution, yielding a larger precursor protein, and is cleaved once the precursor...

Research based on single-case designs (SCD) are frequently utilized in educational settings to evaluate the effect of an intervention on student behavior. Visual analysis is the primary method of evaluation of SCD, despite research noting concerns regarding reliability of the procedure. Recent research suggests that characteristics of the graphic display may contribute to poor reliability and overestimation of intervention effects. This study investigated the effect of increasing or decreasing the data points per x- to y-axis ratio (DPPXYR) on rater evaluations of functional relation and effect size in SCD data sets. Twenty-nine individuals (58.6% male) with experience in SCD were asked to evaluate 40 multiple baseline data sets. Two data sets reporting null, small, moderate, and large intervention effects (8 total) were modified by manipulating the ratio of the x- to y-axis (5 variations), resulting in 40 total graphs. Results indicate that raters scored effects as larger as the DPPXYR decreased. Additionally, a 2-way within-subjects analysis of variance (ANOVA) revealed a significant main effect of DPPXYR manipulation on effect size rating, F(2.11, 58.98) = 58.05, p < .001, η2 = .675, and an interaction between DPPXYR manipulation and magnitude of effect, F(6.71, 187.78) = 11.45, p < .001, η2 = .29. Overall, results of the study indicate researchers and practitioners should maintain a DPPXYR of .14 or larger in the interest of more conservative effect size judgments. (PsycINFO Database Record