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The localization of proteins to particular intracellular compartments often regulates their functions. Zyxin is a LIM protein found prominently at sites of cell adhesion, faintly in leading lamellipodia, and transiently in cell nuclei. Here we have performed a domain analysis to identify regions in zyxin that are responsible for targeting it to different subcellular locations. The N-terminal proline-rich region of zyxin, which harbors binding sites for alpha-actinin and members of the Ena/VASP family, concentrates in lamellipodial extensions and weakly in focal adhesions. The LIM region of zyxin displays robust targeting to focal adhesions. When overexpressed in cells, the LIM region of zyxin causes displacement of endogenous zyxin from focal adhesions. Upon mislocalization of full-length zyxin, at least one member of the Ena/VASP family is also displaced, and the organization of the actin cytoskeleton is perturbed. Zyxin also has the capacity to shuttle between the nucleus and focal adhesion sites. When nuclear export is inhibited, zyxin accumulates in cell nuclei. The nuclear accumulation of zyxin occurs asynchronously with approximately half of the cells exhibiting nuclear localization of zyxin within 2.3 h of initiating leptomycin B treatment. Our results provide insight into the functions of different zyxin domains.

Zyxin contains a proline-rich N-terminal domain that is similar to the C-terminal domain in the ActA protein of the bacteria, Listeria monocytogenes. We screened the entire amino acid sequence of human zyxin for Mena-interacting peptides and found that, as with ActA, proline-rich sequences were the sole zyxin sequences capable of binding to Ena/vasodilator-stimulated phosphoprotein (VASP) family members in vitro. From this information, we tested zyxin mutants in which the proline-rich sequences were altered. The reduction in Mena/VASP binding was confirmed by peptide tests, immunoprecipitation, and ectopic expression of zyxin variants at the surface of mitochondria. By transfection assays we showed that zyxin interaction with Mena/VASP in vivo enhances the production of actin-rich structures at the apical surface of cells. Microinjection into cells of peptides corresponding to the first proline-rich sequence of zyxin caused the loss of Mena/VASP from focal contacts. Furthermore, these peptides reduced the degree of spreading of cells replated after trypsinization. We conclude that zyxin and proteins that harbor similar proline-rich repeats contribute to the positioning of Mena/VASP proteins. The positioning of Ena/VASP family members appears to be important when the actin cytoskeleton is reorganized, such as during spreading.

Villin is an actin-binding protein of the intestinal brush border that bundles, nucleates, caps, and severs actin in a Ca(2+)-dependent manner in vitro. Villin induces the growth of microvilli in transfected cells, an activity that requires a carboxyl-terminally located KKEK motif. By combining cell transfection and biochemical assays, we show that the capacity of villin to induce growth of microvilli in cells correlates with its ability to bundle F-actin in vitro but not with its nucleating activity. In agreement with its importance for microfilament bundling in cells, the KKEK motif of the carboxyl-terminal F-actin-binding site is crucial for bundling in vitro. In addition, substitutions of basic residues in a second site, located in the amino-terminal portion of villin, impaired its activity in cells and reduced its binding to F-actin in the absence of Ca(2+) as well as its bundling and severing activities in vitro. Altogether, these findings suggest that villin participates in the organization and stabilization of the brush border core bundle but does not initiate its assembly by nucleation of actin filaments.

Tyrosine sulfation is a post-translational modification in the trans Golgi that has been found in all animal species studied. In the preceding paper (Baeuerle, P. A., Lottspeich, F., and Huttner, W. B. (1988) J. Biol. Chem. 263, 14925-14929), we have identified the site of tyrosine sulfation in an insect secretory protein, yolk protein 2 (YP2) of Drosophila melanogaster. In the present report, tyrosine sulfation of this protein was examined after expression in a heterologous mammalian cell system. Mouse fibroblasts, transfected with Drosophila YP2 genomic DNA inserted into the eucaryotic expression vector pSV2, secreted the fly protein in sulfated form. Analyses of Drosophila YP2 produced by the mouse cells showed that the features of sulfation of this protein were identical to those previously determined for YP2 isolated from flies. YP2 secreted from mouse fibroblasts was found to be exclusively sulfated on tyrosine residues. The stoichiometry of tyrosine sulfation was approximately 1 mol of sulfate/mol of YP2. Sulfate was linked to the same tyrosine residue as in YP2 isolated from flies, tyrosine 172. These results show that essential parameters of the tyrosine sulfation reaction are very similar in insects and mammals and thus highly conserved in evolution.