Loss of HMW1 and HMW3 in noncytadhering mutants of
            <i>Mycoplasma pneumoniae</i>
             occurs post-translationally

Popham, Phillip L.; Hahn, Tae-Wook; Krebes, K.; Krause, Duncan C.

doi:10.1073/pnas.94.25.13979

Cited by 55 publications

(87 citation statements)

References 33 publications

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“…Domain II (residues 171 to 522; black box [this boundary has been revised from that in reference 4 upon closer examination]) is the acidic, proline-rich (APR) domain common to several M. pneumoniae cytoskeletal proteins (see text). Domain III (the remainder of HMW1; medium gray box) includes a C-terminal domain postulated to be involved in targeting for proteolytic degradation (25). The dark gray box within domain I represents the location of the EAGR box (residues 106 to 136; see Discussion and Fig.…”

Section: Methodsmentioning

confidence: 99%

“…(25). After the cells were washed once with HBSS containing 1 mM methionine and three times with PBS, the pellets from each flask were suspended in 2 ml of PBS.…”

Section: Methodsmentioning

confidence: 99%

“…35 S]methionine as described previously (25). Samples were split three ways in fresh Hayflick medium containing 1 mM unlabeled methionine.…”

Section: Methodsmentioning

confidence: 99%

“…After the cells were washed once with HBSS containing 1 mM methionine and three times with PBS, the pellets from each flask were suspended in 2 ml of PBS. Samples were split, with part being immediately subjected to immunoprecipitation (whole-cell radioimmunoprecipitation [RIP]) (4) and part being osmotically lysed as described above before immunoprecipitation (lysate RIP) (25). Antibodies used were anti-HMW1 (C-terminal domain) and anti-FtsH (predicted surface loop).…”

Section: Methodsmentioning

confidence: 99%

“…Studies of the M6 mutant, in which HMW1 is absent and the cytadherence-accessory protein P30 is truncated (22), indicate that HMW1 is required for proper attachment organelle structure and function (9). In another mutant, designated I-2 (18), proteolytic turnover of HMW1 and several other proteins is accelerated (25), correlating with the absence of the cytadherence-accessory protein HMW2 (6,18,19). The accelerated turnover of HMW1 requires its C-terminal domain, as the absence of that region renders recombinant HMW1 stable in mutant I-2 (9).…”

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confidence: 99%

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Stability of Mycoplasma pneumoniae Cytadherence-Accessory Protein HMW1 Correlates with Its Association with the Triton Shell

et al. 2001

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Mycoplasma pneumoniae adsorbs to host respiratory epithelium primarily by its attachment organelle, the proper function of which depends upon mycoplasma adhesin and cytoskeletal proteins. Among the latter are the cytadherence-associated proteins HMW1 and HMW2, whose specific roles in this process are unknown. In the M. pneumoniae cytadherence mutant I-2, loss of HMW2 results in accelerated turnover of HMW1 and other cytadherence-accessory proteins, probably by proteolysis. However, both the mechanism of degradation and the means by which these proteins are rendered susceptible to it are not understood. In this study, we addressed whether HMW1 degradation is a function of its presence among specific subcellular fractions and established that HMW1 is a peripheral membrane protein that is antibody accessible on the outer surfaces of both wild-type and mutant I-2 M. pneumoniae but to a considerably lesser extent in the mutant. Quantitation of HMW1 in Triton X-100-fractionated extracts from cells pulse-labeled with [35 S]methionine indicated that HMW1 is synthesized in a Triton X-100-soluble form that exists in equilibrium with an insoluble (cytoskeletal) form. Pulse-chase analysis demonstrated that over time, HMW1 becomes stabilized in the cytoskeletal fraction and associated with the cell surface in wild-type M. pneumoniae. The less efficient transition to the cytoskeleton and mycoplasma cell surface in mutant I-2 leads to accelerated degradation of HMW1. These data suggest a role for HMW2 in promoting export of HMW1 to the cell surface, where it is stable and fully functional.Adherence of the human pathogen Mycoplasma pneumoniae to host respiratory epithelial cells constitutes a critical step in the pathway leading to tracheobronchitis and atypical (walking) pneumonia. A polar extension of the mycoplasma cell membrane, the terminal organelle, is the major site of attachment and contains proteins responsible both directly and indirectly for attachment. Although the M. pneumoniae adhesin P1 mediates receptor binding (1, 5, 13), Triton X-100 (TX)-insoluble (hereafter referred to as triton shell or cytoskeletal) cytadherence-accessory proteins are required for both the proper formation of the attachment organelle and the localization of P1 to the attachment organelle (reviewed in reference 17). Loss of some of these proteins results in the failure to accumulate P1 at the attachment organelle and confers irregular cell shapes (9,21,34,35), though the means by which this is manifested is unknown. Therefore, characterization of the biochemical properties and the order of assembly of the cytadherence-accessory proteins is necessary for a fuller understanding of both the regulation of adherence and the structure and formation of the attachment organelle.The cytadherence-accessory protein HMW1 is a 112-kDa phosphoprotein (3, 4) that is concentrated along mycoplasma cell filaments, including the attachment organelle, as revealed by immunoelectron microscopic analyses (34). HMW1 has a modular structure (Fig. 1), including a l...

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Section: Methodsmentioning

confidence: 99%

“…(25). After the cells were washed once with HBSS containing 1 mM methionine and three times with PBS, the pellets from each flask were suspended in 2 ml of PBS.…”

Section: Methodsmentioning

confidence: 99%

“…35 S]methionine as described previously (25). Samples were split three ways in fresh Hayflick medium containing 1 mM unlabeled methionine.…”

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

mentioning

confidence: 99%

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Stability of Mycoplasma pneumoniae Cytadherence-Accessory Protein HMW1 Correlates with Its Association with the Triton Shell

et al. 2001

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The proteome of Mycoplasma pneumoniae, a supposedly “simple” cell

Catrein

Herrmann

2011

Proteomics

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This review covers progress in proteome research on Mycoplasma pneumoniae made over the last 5 years. This bacterium is one of the smallest known self-replicating bacteria. With fewer than 700 proposed proteins, it is well suited to a comprehensive proteome analysis. While all of the proposed genes are transcribed, thus far 620 proteins, about 90% of the predicted proteome, have been identified experimentally. To study the proteome organization of M. pneumoniae, 178 soluble protein complexes were isolated under non-denaturing conditions by tandem affinity chromatography and their composition determined by SDS-PAGE and mass spectrometry. The 62 homomultimeric and 116 heteromultimeric protein complexes could be classified according to 12 different COG functional categories. The complexes interacted with each other to some extent, forming larger assemblies. Protein complexes that were large enough and had specific structures (e.g. ribosomes or DNA-dependent RNA polymerase) were visible and countable in their natural environment by cryo-electron tomography. In addition to characterization of the soluble complexes, the analysis of the Triton X-100 insoluble fraction has a major role in the elucidation of the cytoskeleton-like structure, because by analogy with eukaryotic cells, almost all of the structural proteins involved in its formation, and enriched sub-cellular structures, can be found in this fraction.

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