Proteins complexed to the P1 adhesin of Mycoplasma pneumoniae

Adherence of Mycoplasma pneumoniae to host cells requires several mycoplasm al membrane proteins and cytoskeleton-like proteins in addition to the adhe sin P1, a transmembrane protein of 170 kDa. To analyse interactions of the P1 adhesin with other membrane proteins or with cytoskeleton-like proteins, cross-linking studies were performed in vivo using the permeant reagent pa raformaldehyde. The cross-linked protein complex was isolated by immunoaffi nity chromatography, and proteins complexed to the P1 protein were identifi ed by immunoblot analysis followed by high mass accuracy tryptic peptide ma pping using matrix-assisted laser desorption/ionization mass spectrometry ( MALDI MS). In addition to the P1 protein and a truncated form of the same p rotein, the adhesin-related 30 kDa protein, two membrane proteins of 40 and 90 kDa, the cytoskeleton-associated 65 kDa protein and two cytoskeleton-fo rming proteins. HMW1 and HMW3, were found to be components of the isolated protein complex. Furthermore, the cross-linked complex contained the chaper one DnaK and the E1 alpha subunit of pyruvate dehydrogenase, In summary, it was shown that cytadherence-associated membrane proteins are located in cl ose proximity to cytoskeleton-like proteins, suggesting a functional intera ction between membrane and cytoskeleton-like proteins. DnaK might be involv ed in translocation of proteins from the cytoplasm to the membrane and pyru vate dehydrogenase might be a structural protein of the attachment organell e.