Toc34, a 34-kDa integral membrane protein, is a member of the Toc (translocon at the outer-envelope membrane of chloroplasts) complex, which associates with precursor proteins during protein transport across the chloroplast outer membrane. Here we report the 2.0 A resolution crystal structure of the cytosolic part of pea Toc34 in complex with GDP and Mg2+. In the crystal, Toc34 molecules exist as dimers with features resembling those found in a small GTPase in complex with a GTPase activating protein (GAP). However, gel filtration experiments revealed that dimeric and monomeric forms of Toc34 coexisted in phosphate saline buffer solution at pH 7.2. Mutation of Arg 128, an essential residue for dimerization, to an Ala residue led to the formation of an exclusively monomeric species whose GTPase activity is significantly reduced compared to that of wild type Toc34. These results, together with a number of structural features unique to Toc34, suggest that each monomer acts as a GAP on the other interacting monomer.

Toc34 is a member of the outer membrane translocon complex that mediates the initial stage of protein import into chloroplasts. Toc34, like most outer membrane proteins, is synthesized in the cytosol at its mature size without a cleavable transit peptide. The majority of outer membrane proteins do not require thermolysinsensitive components on the chloroplastic surface or ATP for their insertion into the outer membrane. However, different results have been obtained concerning the factors required for Toc34 insertion into the outer membrane. Using an Arabidopsis homologue of pea Toc34, atToc34, we show that the insertion of atToc34 was greatly reduced by thermolysin pretreatment of chloroplasts as assayed either by protease digestion or by alkaline extraction. The insertion was also dependent on the presence of ATP or GTP. A mutant of atToc34 with the GTP-binding domain deleted still required ATP for optimal insertion, indicating that ATP was used by other protein components in the import system. The ATP-supported insertion was observed even in thermolysin-pretreated chloroplasts, suggesting that the protein component responsible for ATP-stimulated insertion is a different protein from the thermolysinsensitive component that assists atToc34 insertion.Most proteins in chloroplasts are encoded by nuclear genes and post-translationally imported from the cytosol. There appear to be at least two classes of nucleus-encoded chloroplastic proteins, distinguished by the presence or absence of cleavable targeting signals. The first class of proteins are synthesized in the cytosol as higher molecular weight precursor proteins with N-terminal extensions called transit peptides. This class of proteins consists of proteins targeted to the interior of chloroplasts (the inner envelope membrane, the stroma, the thylakoid membrane, and the thylakoid lumen) and two outer membrane proteins (1-4). Transit peptides are necessary and sufficient for the import of these precursor proteins into chloroplasts. The import process is initiated by a binding step that involves specific interaction between the transit peptide and a thermolysin-sensitive protein complex on the chloroplastic surface, followed by translocation of the precursor protein across the envelope. The binding step requires ATP in the 100 M range, and translocation across the envelope requires about 1 mM ATP (5). Several envelope proteins that are associated with precursor proteins in the binding step have been identified (6, 7). These proteins are likely to be components of the machinery responsible for the import of precursor proteins into chloroplasts (8). These proteins are collectively named Toc 1 (translocon of the outer membrane of chloroplasts) or Tic (translocon of the inner membrane of chloroplasts) proteins (9).Members of the second class of nucleus-encoded chloroplastic proteins are synthesized in the cytosol at their mature size without cleavable transit peptides. This class of proteins includes most chloroplastic outer membrane proteins. Much less is known abou...

Most chloroplastic outer envelope membrane proteins are synthesized in the cytosol at their mature size without a cleavable targeting signal. Their insertion into the outer membrane is insensitive to thermolysin pretreatment of chloroplasts and does not require ATP. The insertion has been assumed to be mediated by a spontaneous mechanism or by interaction solely with the lipid components of the outer membrane. However, we show here that insertion of an outer membrane protein requires some trypsin-sensitive and some N-ethylmaleimide-sensitive components of chloroplasts. Association and insertion of the outer membrane protein are saturable and compete with the import of another outer membrane protein. These data suggest that import of chloroplastic outer membrane proteins occurs at specific proteinaceous sites on chloroplasts.

Most chloroplastic outer envelope membrane proteins are synthesized in the cytosol at their mature size without a cleavable targeting signal. Their insertion into the outer membrane is insensitive to thermolysin pretreatment of chloroplasts and does not require ATP. The insertion has been assumed to be mediated by a spontaneous mechanism or by interaction solely with the lipid components of the outer membrane. However, we show here that insertion of an outer membrane protein requires some trypsin-sensitive and some N -ethylmaleimide-sensitive components of chloroplasts. Association and insertion of the outer membrane protein are saturable and compete with the import of another outer membrane protein. These data suggest that import of chloroplastic outer membrane proteins occurs at specific proteinaceous sites on chloroplasts. INTRODUCTIONMost chloroplastic proteins are encoded by the nuclear genome and synthesized in the cytosol. Nuclear-encoded chloroplastic proteins can be divided into roughly two groups on the basis of the presence or absence of cleavable targeting signals. Proteins in the first group are synthesized as higher molecular weight precursors with N-terminal extensions called transit peptides. Import of these precursor proteins into chloroplasts requires ATP and some thermolysin-sensitive receptor proteins on the chloroplastic surface (Cline et al., 1985;Olsen et al., 1989;Theg et al., 1989). This group of proteins includes all proteins destined for the interior of chloroplasts and at least one protein destined for the outer envelope membrane Tranel et al., 1995). The second group of proteins consists of most of the outer membrane proteins identified so far. These outer membrane proteins are synthesized at their mature size in the cytosol without a cleavable transit peptide (Salomon et al., 1990;Li et al., 1991;Ko et al., 1992;Fischer et al., 1994;Kessler et al., 1994;Seedorf et al., 1995;Bolter et al., 1999). The import mechanism for the first group of proteins has been studied extensively, and many protein components of the import machinery have been identified (Chen and Schnell, 1999;Keegstra and Cline, 1999;May and Soll, 1999). In contrast, very little is known about how the outer membrane proteins in the second group are targeted and inserted into the outer membrane.Two unique characteristics mark the import of these outer membrane proteins. For almost all of these proteins, thermolysin pretreatment of chloroplasts and ATP removal have no effect on insertion of the proteins into the outer membrane (Salomon et al., 1990;Li et al., 1991;Ko et al., 1992;Fischer et al., 1994). Because these results suggest that insertion of these proteins does not require any surfaceexposed chloroplastic proteins or energy, their insertion has generally been assumed to be accomplished by a spontaneous mechanism or through interaction with the lipid components of the outer membrane (Bruce, 1998;Keegstra and Cline, 1999).However, the hypotheses of spontaneous insertion or interaction with lipids both have their p...

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Nerve terminals release neurotransmitters from vesicles into the synaptic cleft upon transient increases in intracellular Ca 2+ . This process requires the formation of trans SNARE complexes and is regulated by accessory proteins including nsec1 and COMPLEXIN (CPX). Here we present the crystal structure of neuronal squid Sec1 from squid, which was solved by MAD at 2.4 Å. S-Sec1 folds into a modular arch-shaped three-domain assembly.Comparison of structures of squid s-Sec1 from different crystal forms, and rat nsec1 bound to syntaxin-1a (Misura et al. (2000), Nature 404,[355][356][357][358][359][360][361][362] indicates potential conformational rearrangements in domain 1 and 3. A hinge region between domains 1 and 2 may be involved in binding/release of SYNTAXIN (SX), which may also affect the conformational flexibility of the helical hairpin of domain 3. The release of SX from nsec1 is thought to follow SNARE complex formation. Neuronal SNARE complexes then bind to CPX, which couples neurotransmission to an increase in intracellular calcium. The crystal structure of a squid core CPX/SNARE complex solved by molecular replacement at 2.95 Å resolution shows a helical segment of CPX that binds in anti-parallel fashion to the four-helix bundle of the core SNARE complex. CPX interacts at its C-terminus with SX and SYNAPTOBREVIN around the ionic zero layer of the SNARE complex. We propose that CPX is part of a multi-protein complex that regulates membrane fusion of docked vesicles at a late pre-fusion stage. Toc34, a 34 kDa integral membrane protein, is a member of the Toc (translocon at the outer-envelope membrane of chloroplasts) complex that associates with precursor proteins during protein transport across the chloroplast outer membrane. Here we report the crystal structure of the cytosolic part of pea Toc34 complexed with GDP and Mg at 2.0 Å resolution. In the crystal, the Toc34 molecules exist as dimers with features resembling the ones found in a small GTPase complexed with a GTPase activating protein (GAP). Keywords: NSEC1, SNARES, COMPLEXIN Acta Cryst. (2002). A58 (Supplement), C217Gel-filtration, however, revealed that dimeric and monomeric forms of Toc34 coexisted in phosphate saline buffer (pH 7.2) solution. Mutation of Arg 128, an essential residue for dimerization, to alanine led to the formation of only a monomeric form whose GTPase activity is significantly reduced compared to that of the wild-type Toc34. These results together with a number of structural features unique to Toc34, suggest that each monomer acts as a GAP on the other interacting monomer. I shall describe multiresolution geometry data structures used for the visualization of molecular shape and associated properties (electrostatics, interaction potentials, ...). These geometry data structures while volumetric, allow for smooth piecewise polynomial spline approximations of molecular surfaces, corresponding to level sets of electron density. I shall also describe algorithms for topological, and metric quantification of molecular shape and ...