Stability and subcellular localization of cytadherence-associated protein P65 in Mycoplasma pneumoniae.

The surface protein P65 is a constituent of the Mycoplasma pneumoniae cytoskeleton and is present at reduced levels in mutants lacking the cytadherence accessory protein HMW2. Pulse-chase studies demonstrated that P65 is subject to accelerated turnover in the absence of HMW2. P65 was also less abundant in noncytadhering mutants lacking HMW1 or P30 but was present at wild-type levels in mutants lacking proteins A, B, C, and P1. P65 exhibited a polar localization like that in wild-type M. pneumoniae in all mutants having normal levels of HMW1 and HMW2. Partial or complete loss of these proteins, however, correlated with severe reduction in the P65 level and the inability to localize P65 properly.

Stability of Mycoplasma pneumoniae cytadherence-accessory protein HMW1 correlates with its association with the triton shell.

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.

Structure, function, and assembly of the terminal organelle of Mycoplasma pneumoniae.

Mycoplasmas are cell wall-less bacteria at the low extreme in genome size in the known prokaryote world, and the minimal nature of their genomes is clearly reflected in their metabolic and regulatory austerity. Despite this apparent simplicity, certain species such as Mycoplasma pneumoniae possess a complex terminal organelle that functions in cytadherence, gliding motility, and cell division. The attachment organelle is a membrane-bound extension of the cell and is characterized by an electron-dense core that is part of the mycoplasma cytoskeleton, defined here for working purposes as the protein fraction that remains after extraction with the detergent Triton X-100. This review focuses on the architecture and assembly of the terminal organelle of M. pneumoniae. Characterizing the downstream consequences of defects involving attachment organelle components has made it possible to begin to elucidate the probable sequence of certain events in the biogenesis of this structure.

Overlapping distribution of the 130- and 110-kDa myosin I isoforms on rat liver membranes.

The biochemical and mechanochemical properties and localization of myosin I suggest the involvement of these small members of the myosin superfamily in some aspects of intracellular motility in higher cells. We have determined by quantitative immunoblotting with isoform-specific antibodies that the 130-kDa myosin I (myr 1 gene product) and 110-kDa myosin I (myr 2 gene product) account for 0.5 and 0.4%, respectively, of total rat liver protein. Immunoblot analyses reveal that the 130- and 110-kDa myosins I are found in several purified subcellular fractions from rat liver. The membrane-associated 130-kDa myosin I is found at the highest concentration in the plasma membrane (28 ng/microg plasma membrane protein) followed by the endoplasmic reticulum-like mitochondria-associated membrane fraction (MAM; 10 ng/microg MAM protein), whereas the 110-kDa myosin I is found at the highest concentration in Golgi (50 ng/¿g Golgi protein) followed by plasma membrane (20 ng/microg) and MAM (7 ng/microg). Our analyses indicate that myosin I is peripherally associated with Golgi and MAM and its presence in these fractions is not a consequence of myosin I bound to contaminating actin filaments. Although found in relatively low concentrations in microsomes, because of the abundance of microsomes, in liver most of the membrane-associated myosin I is associated with microsomes. Neither myosin I isoform is detected in purified mitochondria. This is the first quantitative analysis addressing the cellular distribution of these mammalian class I myosins.

Identification of brush border myosin-I in liver and testis.

Brush border myosin-I, or BBMI, constitutes the lateral links that connect in intestinal microvilli the core bundle of actin filaments to the membrane. Although related molecules have been identified in other higher eukaryotic tissues, northern blot analysis has indicated that the distribution of this particular myosin-I isoform is restricted essentially to intestine. Using reverse transcriptase polymerase chain reaction we have identified BBMI in a wide range of tissues including liver and testis. Our results also indicate that in testis the BBMI gene might be alternatively spliced.