A monocytic cell line, termed Mono Mac, was established from peripheral blood of a patient with monoblastic leukemia. Two clones, designated Mono Mac I and Mono Mac 6, were isolated and both were assigned to the monocyte lineage on the basis of morphological, cytochemical and immunological criteria. Most importantly, the clones express NaF-sensitive non-specific-esterase, produce reactive oxygen and stain with MAb My4. Mono Mac 6, in addition, constitutively exhibits phagocytosis of antibody-coated erythrocytes in 80% of the cells and reacts with a panel of MAbs that are specific for mature monocytes, i.e., M42, LeuM3, 63D3, Mo2 and UCHMI. By contrast, the monoblastic cell lines U937 and THP-I are negative for all these markers. Only expression of My4 could be detected after differentiation induced by interferon-gamma (IFN-gamma). Similar treatment of Mono Mac I, however, resulted in staining with all the monocyte-specific MAbs mentioned above, while IFN-gamma treatment of Mono Mac 6 enhanced antigen expression. In addition, the cells showed an increased frequency of multinucleated cells with a rise from 4.8% to 21.9%. Mono Mac 6 appears to be the only one of the cell lines studied to constitutively express phenotypic and functional features of mature monocytes.

The reggie protein family consists of two proteins, reggie-1 and -2, also called flotillins, which are highly ubiquitous and evolutionarily conserved. Both reggies have been shown to be associated with membrane rafts and are involved in various cellular processes such as T-cell activation, phagocytosis and insulin signalling. However, the exact molecular function of these proteins remains to be determined. In addition, the mechanism of membrane association of reggie-1, which does not contain any transmembrane domain, is not known. In this study, we have produced a fusion protein of reggie-1 with enhanced green fluorescent protein and generated targeted substitutions for the inactivation of putative palmitoylation and myristoylation sites. We were able to show that reggie-1 is myristoylated and multiply palmitoylated and that lipid modifications are necessary for membrane association of reggie-1. Overexpression of reggie-1 resulted in the induction of numerous filopodia-like protrusions in various cell lines, suggesting a role for reggie-1 as a signalling protein in actin-dependent processes.

After endocytosis cholera toxin is transported to the endoplasmic reticulum (ER), from where its A1 subunit (CTA1) is assumed to be transferred to the cytosol by an as-yet unknown mechanism. Here, export of CTA1 from the ER to the cytosol was investigated in a cell-free assay using either microsomes loaded with CTA1 by in vitro translation or reconstituted microsomes containing CTA1 purified from V. cholerae. Export of CTA1 from the microsomes was time- and adenosine triphosphate–dependent and required lumenal ER proteins. By coimmunoprecipitation CTA1 was shown to be associated during export with the Sec61p complex, which mediates import of proteins into the ER. Export of CTA1 was inhibited when the Sec61p complexes were blocked by nascent polypeptides arrested during import, demonstrating that the export of CTA1 depended on translocation-competent Sec61p complexes. Export of CTA1 from the reconstituted microsomes indicated the de novo insertion of the toxin into the Sec61p complex from the lumenal side. Our results suggest that Sec61p complex–mediated protein export from the ER is not restricted to ER-associated protein degradation but is also used by bacterial toxins, enabling their entry into the cytosol of the target cell.

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