The cytoskeletal proteins HMW1 and HMW2 are components of the terminal organelle of the cell wall-less bacterium Mycoplasma pneumoniae. HMW1 is required for a tapered, filamentous morphology but exhibits accelerated turnover in the absence of HMW2. Here, we report that a reciprocal dependency exists between HMW1 and HMW2, with HMW2 subject to accelerated turnover with the loss of HMW1. Furthermore, the instability of HMW2 correlated with its failure to localize to the attachment organelle. The C-terminal domain of HMW1 is essential for both function and its accelerated turnover in the absence of HMW2. We constructed HMW1 deletion derivatives lacking portions of this domain and examined each for stability and function. The C-terminal 41 residues were particularly important for proper localization and function in cell morphology and P1 localization, but the entire C-terminal domain was required to stabilize HMW2. The significance of these findings in the context of attachment organelle assembly is considered.The cell wall-less prokaryote Mycoplasma pneumoniae causes tracheobronchitis and primary atypical pneumonia in humans. Mycoplasma adherence to host respiratory epithelium (cytadherence) is mediated largely by the attachment organelle, a polar, membrane-bound, differentiated cell extension that is defined by an electron-dense core (7, 15). Extraction of M. pneumoniae cells with the nonionic detergent Triton X-100 (TX) yields a TX-insoluble fraction that includes this core structure and consists largely of cytoskeletal proteins such as HMW1, HMW2, HMW3, and P65 (Table 1) (6,11,13,20,30,36). These proteins contribute to the architecture of the attachment organelle, including the localization of the major adhesin protein P1 to this structure, but little is known regarding their specific roles or the dynamics of their assembly (19).HMW1 is a peripheral membrane protein on the mycoplasma cell surface but lacks typical secretion signal or transmembrane sequences or significant homology to any proteins found beyond the mycoplasmas (2, 3, 9). HMW1 has been localized to the attachment organelle (30, 32) as well as the trailing filament (32), depending upon the experimental approach. Cytadherence mutant M6 (Table 1) lacks HMW1 and produces the truncated cytadherence-associated protein P30 due to an internal, in-frame deletion (22). This mutant has a striking morphology, lacking the tapered filamentous appearance characteristic of wild-type cells, and fails to localize P1 to the attachment organelle (13). Recombinant wild-type HMW1, but not a truncated derivative lacking the C-terminal 112 amino acids, restores a near-normal morphology and P1 clustering to mutant M6 (13), indicating that HMW1 contributes to the architecture of the attachment organelle and that the C-terminal domain of HMW1 is essential for normal function.HMW2 is a large protein predicted to form dimeric coiled coils, as well as trimeric coiled coils with P28, also a product of the hmw2 gene (5, 21). HMW2 is a component of the attachment organelle and app...

SummaryMycoplasma pneumoniae attachment to host cells requires biogenesis of a functional attachment organelle, including proper localization of the adhesion protein P1 to this structure. Mutations in the hmw2 gene result in the inability to cytadhere, failure to localize P1 to the attachment organelle, altered cell morphology and accelerated turnover of the cytadherence-associated proteins HMW1, HMW3 and P65. The hmw2 gene encodes HMW2 (190 kDa) and P28 (28 kDa), the latter apparently the product of internal translation initiation near the 3 ¢ ¢ ¢ ¢ end of the hmw2 coding region. Transformation of hmw2 mutant I-2 with recombinant wild-type hmw2 restores a wild-type phenotype. In the current study, a severely truncated hmw2 gene with an in frame internal deletion of 80% of the HMW2 coding region that leaves the P28-encoding region intact restored cytadherence to mutant I-2. Transformants produced the expected 38 kDa HMW2 derivative (HMW2 D D D D mid) at levels comparable to that of HMW2 in wild-type cells; like HMW2, HMW2 D D D D mid exhibited marked Triton X-100 insolubility. HMW3, P65 and P28 were fully restored, but not HMW1. These transformants were morphologically similar to wild-type M. pneumoniae but failed to localize P1 to the attachment organelle. Finally, a Cterminally truncated HMW2 derivative was partly Triton X-100 soluble and incapable of restoring HMW1, HMW3 and P65 to wild-type levels. These data are consistent with a model in which the C-terminal domain of HMW2 imparts normal localization to the protein, and this localization itself is required for productive interactions with downstream cytadherenceassociated proteins. Furthermore, these results emphasize the association of HMW1 with P1 clustering.

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