SummaryYersinia enterocolitica cross the intestinal epithelium via translocation through M cells, which are located in the follicle-associated epithelium (FAE) of Peyer's patches (PP). To investigate the molecular basis of this process, studies were performed using a recently developed in vitro model, in which the enterocyte-like cell line Caco-2 and PP lymphocytes are co-cultured in order to establish FAE-like structures including M cells. Here, we demonstrate that Y. enterocolitica does not adhere significantly to the apical membrane of differentiated enterocyte-like Caco-2 cells that express binding sites for Ulex europaeus agglutinin (UEA)-1. In contrast, Y. enterocolitica adhered to, and was internalized by, cells that lacked UEA-1 binding sites and displayed a disorganized brush border. These cells were considered to be converted to M-like cells. Further analysis revealed that part of these cells expressed b1 integrins at their apical surface and, as revealed by comparison of wild-type and mutant strains, interacted with invasin of Y. enterocolitica. Consistently, anti-b1 integrin antibodies significantly inhibited internalization of inv-expressing yersiniae. Experiments with Yersinia mutant strains deficient in YadA or Yop secretion revealed that these virulence factors play a minor role in this process. After internalization, yersiniae were transported within LAMP-1-negative vacuoles to, and released at, the basal surface. Internalization and transport of yersiniae was inhibited by cytochalasin D, suggesting that F-actin assembly is required for this process. These results provide direct evidence that expression of b1 integrins at the apical surface of M cells enables interaction with the invasin of Y. enterocolitica, and thereby initiates internalization and translocation of bacteria.

Yeast adenylate kinase (Aky2p, Adk1p) occurs simultaneously in cytoplasm and mitochondrial intermembrane space. It has no cleavable mitochondrial targeting sequence, and the signal for mitochondrial import and submitochondrial sorting is largely unknown. The extreme N terminus of Aky2p is able to direct cytoplasmic passengers to mitochondria. However, an Aky2 mutant lacking this sequence is imported with about the same efficiency as the wild type. To identify possible importrelevant information in the interior, parts of Aky2p were exchanged by homologous in vitro recombination for the respective segments of the purely cytoplasmic isozyme, Ura6p. Import studies revealed an internal region of about 40 amino acids, which was sufficient to direct the chimera to mitochondria but not for correct submitochondrial sorting. The respective Ura6p hybrid was arrested in the mitochondrial membrane at a position where it was inaccessible to protease but was released by alkaline extraction, suggesting that it had entered an import channel and passed the initial steps of recognition and uptake. Site-specific mutations within the presumptive address-specifying segment identified the amphipathic helix 5. A Ura6 mutant protein in which helix 5 had been replaced with the respective sequence from Aky2p was imported, and this address sequence cooperates with the N terminus in the respective double mutant in a synergistic fashion.