The outer membrane protein PulD (secretin) of Klebsiella oxytoca is required for transport of pullulanase across this membrane. We have purified a multimeric PulD complex from an Escherichia coli strain expressing all the proteins involved in pullulanase secretion. The outer membrane-anchored lipoprotein PulS was found to copurify with PulD. The molar ratio of the two proteins is close to 1:1, and the size of the complex is Ϸ1 MDa. Scanning transmission electron and cryo-electron microscopy analyses showed that the purified complex is a cylindrical structure having a central cavity of Ϸ7.6 nm and peripheral radial spokes. Fusion of proteoliposomes containing the purified complex with a planar lipid bilayer resulted in the appearance of small, voltage-activated, ion-conducting channels. We conclude that the central cavity seen in the electron microscope is part of a large gated channel and propose that the observed current f luctuations correspond to voltage-induced, relatively minor displacements of domains in the purified complex rather than to a complete opening of the secretin channel.

Lon (or La) is a soluble, homooligomeric ATP-dependent protease. Mass determination and cryoelectron microscopy of pure mitochondrial Lon from Saccharomyces cerevisiae identify Lon as a f lexible ring-shaped heptamer. In the presence of ATP or 5-adenylylimidodiphosphate, most of the rings are symmetric and resemble other ATP-driven machines that mediate folding and degradation of proteins. In the absence of nucleotides, most of the rings are distorted, with two adjacent subunits forming leg-like protrusions. These results suggest that asymmetric conformational changes serve to power processive unfolding and translocation of substrates to the active site of the Lon protease.Lon (or La) is a highly conserved ATP-dependent protease found in archaea, eubacteria, and mitochondria (1-5). In mitochondria of the yeast Saccharomyces cerevisiae, ATPdependent proteases not only degrade abnormal proteins (6-8) but also stabilize the mitochondrial genome, regulate mitochondrial gene expression, and facilitate the assembly of oligomeric protein complexes (6, 7, 9-11). The proposed chaperone-like activity of Lon and other ATP-dependent proteases may explain the energy requirement of these enzymes. Their ATPase activity is not required for peptide bond hydrolysis per se but, rather, for unfolding or remodeling target substrates (4,5,(12)(13)(14)(15)(16).The ATP-dependent proteases ClpAP, HslUV, and the proteasome are all ring-shaped structures with 6-or 7-fold symmetry that resemble the chaperonin . This structural similarity suggests that ATP-dependent protein folding and ATP-dependent protein degradation are mechanistically related. ClpAP, HslUV, and the proteasome are two-component ATPase-protease complexes; homooligomeric Lon is unique in that its ATP-dependent chaperone-like function and its proteolytic function are combined within a single subunit. Lon is thus an attractive model to understand structure-function relationships in the more complex ATPdependent proteases. Here, we describe the structure of the Lon (Pim1p) protease from S. cerevisiae mitochondria. MATERIALS AND METHODSPurification. Active Lon carrying six C-terminal histidine residues was overexpressed in yeast, was rapidly purified to homogeneity from a isolated mitochondrial extract on a Ni 2ϩ -NTA column, and purified to homogeneity by gel filtration on a Superose 6 column as described (11); however, where indicated, 1 mM ATP or 1 mM 5Ј-adenylylimidodiphosphate (AMP-PNP) were present during solubilization and purification.Analytical Ultracentrifugation. Purified Lon (1 mg͞ml) was analyzed by analytical ultracentrifugation in 20 mM Hepes (pH 8.0), 150 mM NaCl, and 10% (wt͞vol) glycerol either in the absence or presence of 1 mM ATP. Sedimentation velocity and sedimentation equilibrium determinations were carried out in a Beckman Coulter Model E analytical ultracentrifuge equipped with interference and schlieren optics and in a Beckman Model XLA ultracentrifuge equipped with absorption optics. Sedimentation velocity was performed at 40,000 rpm at 20°C...

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The structures of the DnaB helicase and its complex with DnaC reveal some interesting structural features relevant to helicase function and to the assembly of the two-protein complex. The results presented here provide a basis for a more complete understanding of the structure and function of these important proteins.

The N-ethylmaleimide-sensitive fusion protein (NSF) is an ATPase that plays an essential role in intracellular membrane trafficking. Previous reports have concluded that NSF forms either a tetramer or a trimer in solution, and that assembly of the oligomer is essential for efficient activity in membrane transport reactions. However, in recent electron microscopic analyses NSF appears as a hexagonal cylinder similar in size to related ATPases known to be hexamers. We have therefore reevaluated NSF's oligomeric state using a variety of quantitative biophysical techniques. Sedimentation equilibrium and sedimentation velocity analytical ultracentrifugation, transmission electron microscopy with rotational image analysis, scanning transmission electron microscopy, and multiangle light scattering all demonstrate that, in the presence of nucleotide, NSF is predominantly a hexamer. Sedimentation equilibrium results further suggest that the NSF hexamer is held together by oligomerization of its D2 domains. The sedimentation coefficient, s 20,w 0 , of 13.4 (؎0.1) S indicates that NSF has unusual hydrodynamic characteristics that cannot be solely explained by its shape. The demonstration that NSF is a hexameric oligomer highlights structural similarities between it and several related ATPases which act by switching the conformational states of their protein substrates in order to activate them for subsequent reactions.