Arabidopsis dynamin-like 2 that binds specifically to phosphatidylinositol 4-phosphate assembles into a high-molecular weight complex in vivo and in vitro.

Arabadopsis dynamin-like (ADL) 2, a member of the high-molecular weight (M(r)) dynamin family found in Arabidopsis, has been shown to be targeted to the plastid. In the chloroplast, most of the ADL2 was present in the fraction containing the envelope membranes when analyzed by suborganellar fractionation. Sucrose gradient and gel filtration experiments showed that when associated with membranes, ADL2 existed as a high-M(r) complex, whereas the soluble form existed as a monomer. The recombinant ADL2 expressed in Escherichia coli was present as a high-M(r) form and showed higher GTPase activity at a low NaCl concentration, whereas ADL2 existed as a low-M(r) form with a low level of GTPase activity at a high NaCl concentration. Electron microscopy studies revealed that the purified recombinant ADL2 formed spiral-coiled structures or rings. In the presence of guanosine-5'-O-(3-thio)triphosphate, these structures were transformed into a long rod structure. In contrast, in the presence of GDP, these structures disassembled into oligomers that were shown to be tetramer with 4-fold symmetry. Finally, a lipid-binding assay revealed that recombinant ADL2 purified from E. coli bound specifically to phosphatidylinositol 4-phosphate. Together, these results demonstrated that the biochemical properties of ADL2 were very similar to those of dynamin and other related proteins. Based on this similarity, we propose that ADL2 may be involved in vesicle formation at the chloroplast envelope membrane.

A new dynamin-like protein, ADL6, is involved in trafficking from the trans-Golgi network to the central vacuole in Arabidopsis.

Dynamin, a high-molecular-weight GTPase, plays a critical role in vesicle formation at the plasma membrane during endocytosis in animal cells. Here we report the identification of a new dynamin homolog in Arabidopsis named Arabidopsis dynamin-like 6 (ADL6). ADL6 is quite similar to dynamin I in its structural organization: a conserved GTPase domain at the N terminus, a pleckstrin homology domain at the center, and a Pro-rich motif at the C terminus. In the cell, a majority of ADL6 is associated with membranes. Immunohistochemistry and in vivo targeting experiments revealed that ADL6 is localized to the Golgi apparatus. Expression of the dominant negative mutant ADL6[K51E] in Arabidopsis protoplasts inhibited trafficking of cargo proteins destined for the lytic vacuole and caused them to accumulate at the trans-Golgi network. In contrast, expression of ADL6[K51E] did not affect trafficking of a cargo protein, H(+)-ATPase:green fluorescent protein, destined for the plasma membrane. These results suggest that ADL6 is involved in vesicle formation for vacuolar trafficking at the trans-Golgi network but not for trafficking to the plasma membrane in plant cells.

Heptameric ring structure of the heat-shock protein ClpB, a protein-activated ATPase in Escherichia coli.

The heat-shock protein ClpB is a protein-activated ATPase that is essential for survival of Escherichia coli at high temperatures. ClpB has also recently been suggested to function as a chaperone in reactivation of aggregated proteins. In addition, the clpB gene has been shown to contain two translational initiation sites and therefore encode two polypeptides of different size. To determine the structural organization of ClpB, the ClpB proteins were subjected to chemical cross-linking analysis and electron microscopy. The average images of the ClpB proteins with end-on orientation revealed a seven-membered, ring-shaped structure with a central cavity. Their side-on view showed a two-layered structure with an equal distribution of mass across the equatorial plane of the complex. Since the ClpB subunit has two large regions containing consensus sequences for nucleotide binding, each layer of the ClpB heptamer appears to represent the side projection of one of the major domains arranged on a ring. In the absence of salt and ATP, the ClpB proteins showed a high tendency to form a heptamer. However, they dissociated into various species of oligomers with smaller sizes, depending on salt concentration. Above 0.2 M NaCl, the ClpB proteins behaved most likely as a monomer in the absence of ATP, but assembled into a heptamer in its presence. Furthermore, mutations of the first ATP-binding site, but not the second site, prevented the ATP-dependent oligomerization of the ClpB proteins in the presence of 0.3 M NaCl. These results indicate that ClpB has a heptameric ring-shaped structure with a central cavity and this structural organization requires ATP binding to the first nucleotide-binding site localized to the N-terminal half of the ATPase.

Lamellar stacking in three-dimensional crystals of Ca(2+)-ATPase from sarcoplasmic reticulum.

Electron microscopy of multilamellar crystals of CA(2+)-ATPase currently offers the best opportunity for obtaining a high-resolution structure of this ATP-driven ion pump. Under certain conditions small, wormlike crystals are formed and provide views parallel to the lamellar plane, from which parameters of lamellar stacking can be directly measured. Assuming that molecular packing is the same, data from these views could supplement those obtained by tilting large, thin platelike crystals. However, we were surprised to discover that the lamellar spacing was variable and depended on the amount of glycerol present during crystallization (20% versus 5%). Projection maps (h,0,l) from these womklike crystals suggest different molecular contacts that give rise to the different lamellar spacings. Based on an orthogonal projection map (h,k,0) from collapsed, wormlike crystals and on x-ray powder patterns, we conclude that molecular packing within the lamellar plane is the same as that in thin, platelike crystals and is unaffected by glycerol. Finally, the orientation of molecules in the lamellar plane was characterized from freeze-dried, shadowed crystals. Comparing the profile of molecules in these multilamellar crystals with that previously observed in helical tubes induced by vanadate gives structural evidence of the conformational change that accompanies binding of calcium of Ca(2+)-ATPase.