No abstract

Caveolin-1 is normally localized in plasma membrane caveolae and the Golgi apparatus in mammalian cells. We found three treatments that redirected the protein to lipid storage droplets, identified by staining with the lipophilic dye Nile red and the marker protein ADRP. Caveolin-1 was targeted to the droplets when linked to the ER-retrieval sequence, KKSL, generating Cav–KKSL. Cav–ΔN2, an internal deletion mutant, also accumulated in the droplets, as well as in a Golgi-like structure. Third, incubation of cells with brefeldin A caused caveolin-1 to accumulate in the droplets. This localization persisted after drug washout, showing that caveolin-1 was transported out of the droplets slowly or not at all. Some overexpressed caveolin-2 was also present in lipid droplets. Experimental reduction of cellular cholesteryl ester by 80% did not prevent targeting of Cav–KKSL to the droplets. Cav–KKSL expression did not grossly alter cellular triacylglyceride or cholesteryl levels, although droplet morphology was affected in some cells. These data suggest that accumulation of caveolin-1 to unusually high levels in the ER causes targeting to lipid droplets, and that mechanisms must exist to ensure the rapid exit of newly synthesized caveolin-1 from the ER to avoid this fate.

Abstract. Recent work has demonstrated that p5(~ ok, a member of the Src family of protein tyrosine kinases (PTKs), is modified by palmitoylation of a cysteine residue(s) within the first 10 amino acids of the protein (in addition to amino-terminal myristoylation that is a common modification of the Src family of PTKs). This is now extended to three other members of this family by showing incorporation of [3H]palmitate into p59~, p55f~, and p56 h~, but not into p60 ~. The [3H]palmitate was released by treatment with neutral hydroxylamine, indicating a thioester linkage to the protein. Individual replacement of the two cysteine residues within the first 10 amino acids of p59 fyn and p5(~ ~ with serine indicated that Cys 3 was the major determinant of palmitoylation, as well as association of the PTK with glycosyl-phosphatidylinositol-anchored proteins. Introduction of Cys 3 into p60 ~ led to its palmitoylation, p59 ~" but not p60 s~ partitioned into Triton-insoluble complexes that contain caveolae, microinvaginations of the plasma membrane. Mapping of the requirement for partitioning into caveolae demonstrated that the amino-terminal sequence MetGly-Cys is both necessary and sufficient within the context of a Src family PTK to confer localization into caveolae. Palmitoylation of this motif in p59 fyn also modestly increased its overall avidity for membranes. These results highlight the role of the amino-terminal motif Met-Gly-Cys in determining the structure and properties of members of the Src family of PTKs.T HE Src family of protein tyrosine kinases (PTKs) 1 is comprised of nine members, whose prototype p60 V-~ was first discovered as the transforming gene of Rous sarcoma virus and later found to have as its normal cellular counterpart the protooncogene p60 ¢-~ (3). Many of the members of the Src family are involved in signal transduction and cell activation, with p5~ ~k and p59 fyn being well analyzed examples. Several lines of evidence have implicated p5~ °k and p59 fy* in lymphocyte activation through the T cell antigen receptor (17). These results correlate with the biochemical findings of a direct association between p59 fyn and the CD3/~" chain complex of the T cell receptor (32,42) and between p5(~ ok and the coreceptors CIM and CD8 (31,43).Activation of T cells also occurs after crosslinking of glycosyl-phosphatidylinositol (GPI)-anchored membrane proteins (21, 28). Analysis of the mechanism of lymphocyte activation in this case has led to new insights into the features of GPI-anchored proteins and their role in the cell. The GPI Address all correspondence to Douglas M. Lublin,

Cross-linking of glycosyl-phosphatidylinositol (GPI)-anchored membrane proteins on T cells can trigger cell activation. We and others have shown an association between GPI-anchored proteins and the protein tymsine kinases (PTKs) p56kk and p5905", suggesting a pathway for signaling through GPI-anchored proteins. Studies of decay-accelerating factor (DAF) or CD59 in either the C32 cell line or the HeLa cell line transfected with PTK cDNA demonstrated that the GPI-anchored proteins associated noncovalently with p56kk and p590yr' but not with p60Y. Nonmyristylated versions of p56k* and p590y"' also failed to associate with the GPI-anchored proteins. Mutational analysis of the PTK demonstrated that the association with the GPI-anchored proteins mapped to the unique amino-terminal domains of the PTK. A chimeric PTK consisting of the 10 aminoterminal residues of p56kk or p59r"' replacing the corresponding amino acids in p6W' was sufficient for association with DAF, but the converse constructs containing the first 10 amino acids of p60' plus the remainder of p56kk or p59'"' did not associate with DAF. Mutation of cysteine to serine at positions 3 and 6 in p59'" or positions 3 and 5 in p56k! abolished the association of these kinases with DAF. Mutation of serine to cysteine at positions 3 and 6 in p60"" conferred on p6(YFv the ability to associate with DAF. Direct labeling with [3H]palmitate demonstrated palmitylation of this amino-terminal cysteine motif in p56kk. Thus, palmitylation of the amino-terminal cysteine residue(s) together with myristylation of the amino-terminal glycine residue defines important motifs for the association of PTKs with GPI-anchored proteins.

Strains of Escherichia coli persist within the human gut as normal commensals, but are frequent pathogens and can cause recurrent infection. Here we show that, in contrast to E. coli subjected to opsonic interactions stimulated by the host's immune response, E. coli that bind to the macrophage surface exclusively through the bacterial lectin FimH can survive inside the cell following phagocytosis. This viability is largely due to the attenuation of intracellular free-radical release and of phagosome acidification during FimH-mediated internalization, both of which are triggered by antibody-mediated internalization. This different processing of non-opsonized bacteria is supported by morphological evidence of tight-fitting phagosomes compared with looser, antibody-mediated phagosomes. We propose that non-opsonized FimH-expressing E. coli co-opt internalization of lipid-rich microdomains following binding to the FimH receptor, the glycosylphosphatidylinositol-linked protein CD48, because (1) the sterol-binding agents filipin, nystatin and methyl beta-cyclodextrin specifically block FimH-mediated internalization; (2) CD48 and the protein caveolin both accumulate on macrophage membranes surrounding bacteria; and (3) antibodies against CD48 inhibit FimH-mediated internalization. Our findings bring the traditionally extracellular E. coli into the realm of opportunistic intracellular parasitism and suggest how opportunistic infections with FimH-expressing enterobacteria could occur in a setting deprived of opsonizing antibodies.