Actin-depolymerizing factor (ADF)/cofilins are small actin-binding proteins found in all eukaryotes. In vitro, ADF/cofilins promote actin dynamics by depolymerizing and severing actin filaments. However, whether ADF/cofilins contribute to actin dynamics in cells by disassembling "old" actin filaments or by promoting actin filament assembly through their severing activity is a matter of controversy. Analysis of mammalian ADF/cofilins is further complicated by the presence of multiple isoforms, which may contribute to actin dynamics by different mechanisms. We show that two isoforms, ADF and cofilin-1, are expressed in mouse NIH 3T3, B16F1, and Neuro 2A cells. Depleting cofilin-1 and/or ADF by siRNA leads to an accumulation of F-actin and to an increase in cell size. Cofilin-1 and ADF seem to play overlapping roles in cells, because the knockdown phenotype of either protein could be rescued by overexpression of the other one. Cofilin-1 and ADF knockdown cells also had defects in cell motility and cytokinesis, and these defects were most pronounced when both ADF and cofilin-1 were depleted. Fluorescence recovery after photobleaching analysis and studies with an actin monomer-sequestering drug, latrunculin-A, demonstrated that these phenotypes arose from diminished actin filament depolymerization rates. These data suggest that mammalian ADF and cofilin-1 promote cytoskeletal dynamics by depolymerizing actin filaments and that this activity is critical for several processes such as cytokinesis and cell motility.

The actin cytoskeleton plays a central role in many cell biological processes. The structure and dynamics of the actin cytoskeleton are regulated by numerous actin-binding proteins that usually contain one of the few known actin-binding motifs. WH2 domain (WASP homology domain-2) is a V V35 residue actin monomer-binding motif, that is found in many different regulators of the actin cytoskeleton, including the L L-thymosins, ciboulot, WASP (Wiskott Aldrich syndrome protein), verprolin/ WIP (WASP-interacting protein), Srv2/CAP (adenylyl cyclaseassociated protein) and several uncharacterized proteins. The most highly conserved residues in the WH2 domain are important in L L-thymosin's interactions with actin monomers, suggesting that all WH2 domains may interact with actin monomers through similar interfaces. Our sequence database searches did not reveal any WH2 domain-containing proteins in plants. However, we found three classes of these proteins: WASP, Srv2/ CAP and verprolin/WIP in yeast and animals. This suggests that the WH2 domain is an ancient actin monomer-binding motif that existed before the divergence of fungal and animal lineages. ß

Polypeptides targeted to the yeast endoplasmic reticulum (ER) posttranslationally are thought to be kept in the cytoplasm in an unfolded state by Hsp70 chaperones before translocation. We show here that Escherichia coli ␤-lactamase associated with Hsp70, but adopted a native-like conformation before translocation in living Saccharomyces cerevisiae cells. ␤-Lactamase is a globular trypsin-resistant molecule in authentic form. For these studies, it was linked to the C terminus of a yeast polypeptide Hsp150⌬, which conferred posttranslational translocation and provided sites for O-glycosylation. We devised conditions to retard translocation of Hsp150⌬-␤-lactamase. This enabled us to show by protease protection assays that an unglycosylated precursor was associated with the cytoplasmic surface of isolated microsomes, whereas a glycosylated form resided inside the vesicles. Both proteins were trypsin resistant and had similar ␤-lactamase activity and K m values for nitrocefin. The enzymatically active cytoplasmic intermediate could be chased into the ER, followed by secretion of the activity to the medium. Productive folding in the cytoplasm occurred in the absence of disulfide formation, whereas in the ER lumen, proper folding required oxidation of the sulfhydryls. This suggests that the polypeptide was refolded in the ER and consequently, at least partially unfolded for translocation. INTRODUCTIONTranslocation of newly synthesized precursor proteins into the yeast endoplasmic reticulum (ER) occurs cotranslationally or posttranslationally, depending on the hydrophobicity of the signal peptides (Brodsky and Schekman, 1994;Ng et al., 1996;Rapoport et al., 1996). In the cotranslational pathway, the signal recognition particle binds to the signal peptide emerging from the ribosome, translation halts, and the nascent chain-ribosome complex is targeted to the trimeric Sec61 translocon complex (Sec61p, Sbh1p, and Sss1p) embedded in the ER membrane (Panzner et al., 1995). The polypeptide traverses the aqueous translocon channel simultaneously with elongation, apparently in an extended form, whereafter it adopts its native structure in the ER lumen. For instance, in cotranslational translocation of the Escherichia coli Lep protein into canine microsomes, 65 amino acids bridge the ribosomal P site and the luminal surface of the ER membrane (Whitley et al., 1996). In contrast, posttranslational translocation is signal recognition particle independent. Translation of the polypeptide is completed on free ribosomes, whereafter the preprotein traverses the ER membrane via the translocon complex associated with the Sec62-63 complex (Sec62p, Sec63p, Sec71p, and Sec72p;Deshaies and Schekman, 1989;Rothblatt et al., 1989;Deshaies et al., 1991;Feldheim and Schekman, 1994;Panzner et al., 1995). Because the amino acid sequence primarily dictates the three-dimensional structure of proteins, polypeptides could fold in the cytoplasm unless they were prevented from folding. Depletion of two of the four predominant Hsp70 homologues of the yea...

We found recently that ␤-lactamase folds in the yeast cytosol to a native-like, catalytically active, and trypsinresistant conformation, and is thereafter translocated into the ER and secreted to the medium. Previously, it was thought that pre-folded proteins cannot be translocated. Here we have studied in living yeast cells whether ␤-lactamase, a tight globule in authentic form, must be unfolded for ER translocation. A ␤-lactamase mutant (E166A) binds irreversibly benzylpenicillin via Ser 70 in the active site. We fused E166A to the C terminus of a yeast-derived polypeptide having a post-translational signal peptide. In the presence of benzylpenicillin, the E166A fusion protein was not translocated into the endoplasmic reticulum, whereas translocation of the unmutated variant was not affected. The benzylpenicillin-bound protein adhered to the endoplasmic reticulum membrane, where it prevented translocation of BiP, carboxypeptidase Y, and secretory proteins. Although the 321-amino acid-long N-terminal fusion partner adopts no regular secondary structure and should have no constraints for pore penetration, the benzylpenicillin-bound protein remained fully exposed to the cytosol, maintaining its signal peptide. Our data suggest that the ␤-lactamase portion must unfold for translocation, that the unfolding machinery is cytosolic, and that unfolding of the remote C-terminal ␤-lactamase is required for initiation of pore penetration.

We used the rat nerve growth factor receptor ectodomain (NGFRe) and Escherichia coli ss-lactamase to dissect the functions of Saccharomyces cerevisiae BiP/Kar2p in vivo. Both were fused to the Hsp150Delta-polypeptide, which promotes proper folding of heterologous proteins which otherwise are misfolded in the yeast ER. Hsp150Delta-NGFRe and Hsp150Delta-beta-lactamase acquired disulfides and were properly folded and ONcreted to the culture medium. When disulfide formation was prevented by incubating cells with dithiothreitol (DTT), Hsp150Delta-NGFRe remained in the endoplasmic reticulum (ER). The occupancy of an otherwise partially used N-glycosylation site of reduced NGFRe was complete suggesting that, normally, folding and disulfide formation occurred as rapidly as N-glycosylation. Removal of DTT resulted in remarkably rapid disulfide formation and secretion, suggesting only mild conformational distortion of reduced NGFRe. In contrast, reduced Hsp150(Delta)-ss-lactamase was severely misfolded and attained a secretion competent conformation more slowly after reoxidation. When kar2-159 cells were incubated at permissive temperature 24 degrees C with DTT, the reporter proteins were retained in the ER. After shift of the cells to 34 degrees C to inactivate BiP/Kar2p irreversibly, and subsequent removal of DTT, most pre-accumulated Hsp150Delta-NGFRe was rapidly secreted, whereas Hsp150Delta-beta-lactamase was secretion incompetent. Thus, Hsp150Delta-NGFRe did not require BiP/Kar2p for conformational maturation, though translocation was dependent on BiP/Kar2p. Apparently proteins differ in their post-translocational requirements for BiP/Kar2p, indicating that translocation and chaperoning are distinct functions.