[PMC free article] [PubMed] [Google Scholar] 24

[PMC free article] [PubMed] [Google Scholar] 24. Colonization of the respiratory mucosa is mediated in large part by the terminal organelle, a polar, tapered extension of the mycoplasma cell having a high density of receptor-binding proteins (4, 22, 28). The terminal organelle also constitutes the motor in gliding motility (5, 11), and its duplication precedes cell division (5, 12, 32). Ultrastructurally, the terminal organelle is defined by a characteristic electron-dense core consisting of a thick rod and a thin rod oriented longitudinally in parallel and capped by a terminal button at the distal end (4, 16, 17, 39, 41). The core and terminal button are elements of the mycoplasma cytoskeleton (triton shell), a complex network of proteins resistant to extraction with Triton X-100 (TX) (1, 7, 24), much like the cytoskeletal fraction XL765 of eukaryotic cells (18, RICTOR 33, 34). The composition of the triton shell has been examined by using antibody probes (22) and by mass spectrometry (29), but the identities of proteins specific to the electron-dense core are largely unknown, although cores fail to assemble in the absence of cytoskeletal proteins HMW1 and HMW2, both of which localize to the terminal organelle (3, 31, 36). HMW2 is a large protein (1,818 residues) predicted to have a globular N terminus followed by 10 dimeric or trimeric coiled-coil domains interspersed with leucine zipper motifs (23) XL765 (Fig. ?(Fig.1).1). Spontaneously arising mutant I-2 lacks HMW2 due to a frameshift in the corresponding MPN310 open reading frame, which also encodes protein P28 at its 3 end, in the same reading frame encoding HMW2 (6). Mutants C1 and H9 are similar to mutant I-2 but result from Tndisruption of MPN310 (15, 23) (Fig. ?(Fig.1).1). The loss of HMW2 and the inability of these mutants to assemble a core are accompanied by an abnormal morphology, reduced levels of terminal organelle proteins HMW1, HMW3, P24, P28, P41, and P65, failure to localize the major adhesin P1 to the terminal organelle, and the loss of cytadherence (6, 20, 22, 31). Imprecise transposon excision from mutant C1 yielded excision revertant C1R1, having an in-frame deletion in MPN310 that truncates HMW2 and eliminates P28 (6) (Fig. ?(Fig.1).1). Analysis using immunofluorescence (40) or fluorescent protein fusions (3, 19) localizes HMW2 generally to the terminal organelle. Based on its localization, its requirement for core formation, and its deduced length relative to that of the core, we proposed previously that HMW2 is a major component of the electron-dense core and, with P28, may form bundles oriented longitudinally to yield the large rod of the core (3). Open in a separate window FIG. 1. Structural features of the indicated wild-type (WT), engineered, and mutant HMW2 proteins. White boxes, predicted dimeric coiled XL765 coils; black boxes, predicted trimeric coiled coils; dark gray boxes, leucine zipper motifs; arrows, predicted N terminus of P28; black triangles, cysteine residues. The numbers above each diagram correspond to the coiled-coil regions, while the letters below correspond to the leucine zipper motifs. In the present study, we explored further the role of HMW2 in core formation relative to the current model, by which (i) HMW2 is predicted to orient with its N- and C-terminal domains at the ends of the large rod of the core and (ii) mutants producing shorter HMW2 proteins XL765 are expected to have correspondingly shorter cores. We report here the successful localization.