Chemokines are a structurally related family of cytokines that are important for leukocyte trafficking. The C-C chemokine monocyte chemoattractant protein-1 (MCP-1) is a potent monocyte activator in vitro and has been associated with monocytic infiltration in several inflammatory diseases. One C-C chemokine receptor, CCR2, has been identified that mediates in vitro responses to MCP-1 and its close structural homologues. CCR2 has also recently been demonstrated to be a fusion cofactor for several HIV isolates. To investigate the normal physiological function of CCR2, we generated mice with a targeted disruption of the ccr2 gene. Mice deficient for CCR2 developed normally and had no hematopoietic abnormalities. However, ccr2 −/− mice failed to recruit macrophages in an experimental peritoneal inflammation model. In addition, these mice were unable to clear infection by the intracellular bacteria, Listeria monocytogenes. These results suggest that CCR2 has a nonredundant role as a major mediator of macrophage recruitment and host defense against bacterial pathogens and that MCP-1 and other CCR2 ligands are effectors of those functions.

Abstract. When nuclear localization sequences (termed NLS) are placed at the N terminus of cytochrome ca, a mitochondrial inner membrane protein, the resulting hybrid proteins do not assemble into mitochondria when synthesized in the yeast Saccharomyces cerevisiae. Cells lacking mitochondrial cytochrome c~, but expressing the hybrid NLScytochrome c, proteins, are unable to grow on glycerol since the hybrid proteins are associated primarily with the nucleus. A similar hybrid protein with a mutant NLS is transported to and assembled into the mitochondria. To identify proteins that might be involved in recognition of nuclear localization signals, we isolated conditional-lethal mutants (npl, for nuclear protein localization) that missorted NLS-cytochrome c, to the mitochondria, allowing growth on glycerol. The gene corresponding to one complementation group (NPLI) encodes a protein with homology to DnaJ, an Escherichia coli heat shock protein, npU-1 is allelic to sec63, a gene that affects transit of nascent secretory proteins across the endoplasmic reticulum. Rothblatt, J. A., R. J. Deshaies, S. L. Sanders, G. Daum, and R. Schekman. 1989. J. Cell Biol. 109:2641-2652. The npU mutants reported here also weakly affect translocation of preprocarboxypeptidaseY across the ER membrane. A normally nuclear hybrid protein containing a NLS fused to invertase and a nucleolar protein are not localized to the nucleus in np11/sec63 cells at the nonpermissive temperature. Thus, NPLI/SEC63 may act at a very early common step in localization of proteins to the nucleus and the ER. Alternatively, by affecting ER and nuclear envelope assembly, npll may indirectly alter assembly of proteins into the nucleus.ACH organelle in a eukaryotic cell has a distinct set of proteins that are necessary for its specific function. Certain peptides can act as signals to localize proteins to particular organelles such as the ER, the mitochondria (Verner and Schatz, 1988) and the nucleus (Silver and Hall, 1988). Several proteins have been identified that mediate the recognition of ER-destined proteins and their subsequent translocation across or assembly into the ER membrane (Walter and Blobel, 1980;Meyer et al., 1982;Tajima et al., 1986; Wiedrnan et al., 1987). Receptors have been proposed for mitochondrial signal peptides (Pfaller and Neupert, 1987;Pfanner et al., 1987) and recently a receptor for protein import into chloroplasts has been identified (Pain et al., 1988). By analogy, similar components may exist for localization of proteins to the nucleus.Nuclear localization sequences (NLS)' are stretches of amino acids that are capable of redirecting nonnuclear pro-1. Abbreviations used in this paper: DAPI, diamidinophenylindole; EMS, ethyl methanesulfonate; NLS, nuclear localization sequences; preproCPY, preprocarboxypeptidase Y.teins such as/$-galactosidase to the nucleus. When the first 74 amino acids of the yeast DNA binding protein GAL4 are joined to/~-galactosidase, the result is a fusion protein that is found exclusively in the yeast nucleus as...

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Mutations in the SEC63 gene are associated with defects in protein translocation into the endoplasmic reticulum (ER) as well as in nuclear protein localization in Saccharomyces cerevisiae. To identify proteins that might interact and/or function with SEC63p, we cloned a high copy suppressor (HSS1) of the temperature-sensitive lethal phenotype of the sec63-101 mutant. HSS1 is an allele-specific sec63 suppressor that encodes an integral ER membrane glycoprotein of 206 amino acids with the N-terminus in the ER lumen and C-terminal region in the cytoplasm. Haploid strains disrupted for HSS1 are temperature-sensitive for growth and accumulate precursor forms of Kar2p and invertase. The HSS1 null allele is synthetically lethal in combination with mutations affecting ER translocation. We propose that HSSlp is important for ER translocation and interacts with previously identified components of the yeast translocation apparatus. HSS1 is identical to SEC66, which encodes a glycoprotein complexed with SEC62p and SEC63p.

Abstract. Short stretches of amino acids, termed nuclear localization sequences (NLS), can mediate assembly of proteins into the nucleus. Proteins from the yeast, Saccharomyces cerevisiae, have been identified that specifically recognize nuclear localization peptides (Silver, P., I. Sadler, and M. A. Osborne. 1989. J. Cell Biol. 109:983-989). We now further define the role of one of these NLS-binding proteins in nuclear protein localization. The NLS-binding protein of 70-kD molecular mass can be purified from salt extracts of nuclei. Antibodies,raised against the NLS-binding protein localized the protein mainly to the nucleus with minor mounts in the cytoplasm. These antibodies also inhibited the association of NLS-protein conjugates with nuclei. Incubation of nuclei with proteases coupled to agarose removed NLS-binding protein activity. Extracts enriched for NLS-binding proteins can be added back to salt or protease-treated nuclei to restore NLSbinding activity. These results suggest that the first step of nuclear protein import can be reconstituted in vitro.T HE nucleus is characterized by its distinct set of proteins. One model for nuclear protein uptake proposes that it is an active process, triggered by specific interaction of a short stretch of amino acids contained within the transported protein (termed nuclear localization sequence [NLS] 1) and one or more "receptors; perhaps located at the nuclear pore complex. Nuclear localization sequences are found in many nuclear proteins (for examples see Kalderon et al., 1984;Hall et al., 1984;and Silver et al., 1984) and are necessary for protein transit across the nuclear envelope via the nuclear pores (Feldherr et al., 1984; al., 1988;Newmeyer and Forbes, 1988).Specific information for nuclear protein localization resides not only within the transported protein as evidenced by the presence of nuclear localization sequences, but also in the transport apparatus. Evidence for receptors that recognize proteins for nuclear import was first provided by Goldfarb etal. (1986), who cross-linked BSA to the SV40 T-antigen NLS and found that the rate of nuclear uptake for the complex was saturable. Further support for receptor-mediated uptake comes from microinjection of nuclear proteins into animal cells (Lanford et al., 1986;Richardson et al., 1988;Breeuwer and Goldfarb, 1990) and in vitro experiments with nuclei from rat liver (Marldand et al., 1987), Xenopus oocytes (Newmeyer and Forbes, 1988;Newmeyer et al., 1986), and yeast (Kalinich and Douglas, 1989). Nuclei will import SV40 T-antigen and Xenopus nucleoplasrain in a manner that is dependent on the NLS, temperature, and ATP. In the absence of ATP, gold particles coated with the SV40 NLS bind at the pore, but are not imported, sug-1. Abbreviation used in this paper: NLS, nuclear localization sequence. gesting that nuclear localization is at least a two-step process: binding followed by translocation through the pore (Richardson et al., 1988;Newmeyer and Forbes, 1988).Nuclear and cytoplasmic proteins from yeast Lee...