SummaryPhosphatidylinositol (3,4,5)-trisphosphate [PtdIns(3,4,5)P 3 ] is a key regulator of cell signaling that acts by recruiting proteins to the cell membrane, such as at the leading edge during cell migration. Here, we show that PtdIns (3,4,5)P 3 plays a central role in filopodia formation via the binding of myosin-X (Myo10), a potent promoter of filopodia. We found that the second pleckstrin homology domain (Myo10-PH2) of Myo10 specifically binds to PtdIns(3,4,5)P 3 , and that disruption of this binding led to impairment of filopodia and partial re-localization of Myo10 to microtubule-associated Rab7-positive endosomal vesicles. Given that the localization of Myo10 was dynamically restored to filopodia upon reinstatement of PtdIns(3,4,5)P 3 -binding, our results indicate that PtdIns(3,4,5)P 3 binding to the Myo10-PH2 domain is involved in Myo10 trafficking and regulation of filopodia dynamics.

Receptor priming of low-pH-triggered virus entry has been described for an enveloped virus (15). Here we show with major group human rhinoviruses (HRV) and its intercellular adhesion molecule-1 receptor that nonenveloped viruses follow this novel cell entry principle. In vitro the receptor primed HRV for efficient uncoating at mild low pH (5.5 to 6.0). Agents preventing endosomal acidification reduced or blocked rhinovirus cell infection, while nocodazole had no effect on infection of any serotype tested. The entry inhibitory effect of lysosomotropic agents was overcome by exposing cell-internalized HRV to mild low pH (5.5 to 6.0). We therefore conclude that receptor priming of major group HRV must occur in vivo as well. Cooperation of a cellular receptor and low pH in virus uncoating will polarize the exit of the genome to the receptor-bound, membrane-proximal region of the virus particle during acidification of endosomes. This process must be required for efficient penetration of the cellular membrane by viruses.Entry of viruses into host cells requires binding to one or several cell surface receptors and subsequent penetration of the cellular membrane. The penetration or entry process in enveloped viruses occurs by fusion of virus and cell membranes (21), while in nonenveloped viruses the process requires local disruption of the bilayer (3). For most viruses, cell entry is mediated by either receptor (pH independent) or low pH. Recently, however, it was shown that entry of the retrovirus avian leukosis virus (ALV) required both receptor and low pH, providing a novel principle for entry of enveloped viruses (15). Binding of ALV to its cellular receptor converts the viral envelope protein into a metastable form sensitive to low pH so that subsequent virus internalization and endosomal acidification triggers membrane fusion and release of the viral capsid into the cell cytoplasm.Cell entry and receptor recognition have been extensively studied in picornaviruses, a large family of nonenveloped viruses responsible of several human and animal diseases (19). Picornaviruses are constituted by an icosahedral protein capsid built by 60 protomers assembled in 12 pentamers, with a singlestranded RNA genome closely packed inside. Cell entry requires uncoating or exit of the RNA from the capsid, which presumably moves to the cell cytoplasm through a membrane pore generated by hydrophobic capsid polypeptides (3). Receptor-mediated uncoating of picornaviruses at neutral pH was first described for poliovirus (12). Poliovirus does not require a low-pH step or endocytosis for cell entry (8, 16), so multimeric binding of the virus to the receptor must trigger the molecular events leading to virus penetration (18). The poliovirus entry pathway differs from that described for human rhinovirus serotype 2 (HRV2), a member of the minor group of HRV, which bind to receptors belonging to the low-density lipoprotein (LDL) family (10). HRV2 requires both endocytosis and a low-pH step for efficient uncoating and cell entry (1, 2). The LDL...

NaChBac from Bacillus halodurans is a bacterial homologue of mammalian voltage-gated sodium channels. It has been proposed that a NaChBac monomer corresponds to a single domain of the mammalian sodium channel and that, like potassium channels, four monomers form a tetrameric channel. However, to date, although NaChBac has been well-characterized for functional properties by electrophysiological measurements on protein expressed in tissue culture, little information about its structural properties exists because of the difficulties in expressing the protein in large quantities. In this study, we present studies on the overexpression of NaChBac in Escherichia coli, purification of the functional detergent-solubilized channel, its identification as a tetramer, and characterization of its secondary structure, drug binding, and thermal stability. These studies are correlated with a model produced for the protein and provide new insights into the structure-function relationships of this sodium channel.

The NaChBac sodium channel from Bacillus halodurans is a homologue of eukaryotic voltage-gated sodium channels. It can be solubilized in a range of detergents and consists of four identical subunits assembled as a tetramer. Sodium channels are relatively flexible molecules, adopting different conformations in their closed, open and inactivated states. This study aimed to design and construct a mutant version of the NaChBac protein that would insert into membranes and retain its folded conformation, but which would have enhanced stability when subjected to thermal stress. Modelling studies suggested a G219S mutant would have decreased conformational flexibility due to the removal of the glycine hinge around the proposed gating region, thereby imparting increased resistance to unfolding. The mutant expressed in Escherichia coli and purified in the detergent dodecyl maltoside was compared to wildtype NaChBac prepared in a similar manner. The mutant was incorporated into the membrane fraction and had a nearly identical secondary structure to the wildtype protein. When the thermal unfolding of the G219S mutant was examined by circular dichroism spectroscopy, it was shown to not only have a Tm approximately 10 degrees C higher than the wildtype, but also in its unfolded state it retained more ordered helical structure than did the wildtype protein. Hence the G219S mutant was shown to be, as designed, more thermally stable.

Plasma membranes from the green alga Chlamydomonas reinhardtii were purified by differential centrifugation and two‐phase partitioning in an aqueous polymer system. The isolated plasma membranes were virtually free from contaminating chloroplasts, mitochondria, endoplasmic reticulum and Golgi membranes as shown by marker enzyme and pigment analysis. The isolated plasma membranes exhibited vanadate sensitive ATPase activity, indicating the presence of a P‐type ATPase. This was verified by using antibodies against P‐type ATPase from Arabidopsis, which crossreacted with a protein of 109 kDa. The ATPase activity was inhibited to more than 90% by vanadate (Ki= 0.9 μM) but not affected by inhibitors specific for F‐ or V‐type ATPases. demonstrating the purity of the plasma membranes. Mg‐ATP was the substrate, and the rate of ATP‐hydrolysis followed simple Michaelis‐Menten kinetics giving a Km= 0.46 mM. Free Mg2+ stimulated the activity, K1/2= 0.68 mM. Maximal activity was obtained at pH 8. The ATPase activity was latent but stimulated 10 to 20‐fold in the presence of detergents. This indicates that the isolated plasma membrane vesicles were tightly sealed and mostly right‐side‐out, making the ATPase inaccessible to the hydrophilic substrate ATP. In the presence of the Brij 58, the isolated plasma membranes performed ATP dependent H+‐pumping as shown by the optical pH probe acridine orange. H+‐pumping was dependent on the presence of valinomycin and K+ ions and completely abolished by vanadate. Addition of Brij 58 has been shown to produce 100% sealed inside‐out vesicles of plant plasma membranes (Johansson et al. 1995, Plant J. 7: 165–173) and this was also the case for plasma membranes from the green alga Chlamydomonas reinhardtii.