The cGMP phosphodiesterase (PDE) of cattie retinal rod outer segments comprises three types of subunits: the two heavy catalytic ones, PDEa and PDE.8, each around 85 kDa, and the light inhibitory one, PDEy or I (11 kDa). The relative stoichiometry is usually assumed to be 1:1:1. PDE activation in the visual transduction cascade results from removal of the inhibitor by the a subunit of transducin (Ta). The stoichiometric complex Ta-I, separated from activated PDE, has been isolated and characterized. Analyzing now the activated PDE, we find that it still contains some inhibitor and is resolvable into two species, one with 50% of the inhibitor content of the native enzyme and the other totally devoid of it. The same two species are observed upon activation of PDE by very short tryptic proteolysis, which specifically degrades the inhibitor. This leads us to conclude that the composition of the native enzyme is PDEa8-I2. (14) and preserved at -80'C. The thawed pellets were homogenized, illuminated, and then washed in medium salt buffer, to eliminate some minor proteins not relevant to this study. Extraction and "crude" purification were then carried out using the light, nucleotide, and ionic-strength dependence of binding of the species (14). Crude PDE was obtained by low ionic strength extraction of the illuminated membrane pellet and crude transducin was obtained by subsequent extraction of the same pellet after addition of 100 ,uM GTP[yS]. Total extract was obtained by direct low ionic strength extraction of an illuminated membrane pellet in the presence of GTP [yS]. Low salt buffer was 5 mM Hepes/1 mM dithiothreitol, pH 7.5; medium salt buffer was 20 mM Hepes/120 mM KCl/1 mM dithiothreitol, pH 7.5. Rhodopsin concentration in all extraction procedures was 2 mg/ml. GTP [yS] was used at 100 ,uM.Protein Chromatography. Separation and purification of transducin subunits, native PDE, and the various PDE units were performed as described (15), on an ion exchange column (Polyanion SI from Pharmacia) (unfortunately this very efficient column is not commercially available anymore, but we have a stock of them). Elution was obtained by Na2SO4 gradients (from 0 to 600 mM) in a buffer containing 20 mM Hepes, pH 7.5/1 mM MgSO4/5 mM 2-mercaptoethanol. Various gradient programs were used to optimize the separation of the different PDE species (see figure legends). Proteins were eluted from the column (0.5 ml/min) followed by UV absorption. Fractions (250 ,ul) 2424The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

SummaryThe lipopolysaccharides (LPS) of intracellular Proteobacteria such as Brucella , Chlamydia , Legionella and Rickettsia , have properties distinct from enterobacterial LPSs. These properties include deficient LPS induction of host cell activation, low endotoxicity and resistance to macrophage degradation. Together these constitute key virulence mechanisms for intracellular survival and replication. We previously demonstrated that B. abortus LPS captured by macrophages was recycled back to the plasma membrane where it was found associated with macrodomains. Furthermore, this LPS interferes with the MHC class II (MHC-II) presentation of peptides to specific T cell hybridomas. Here, we characterized the Brucella LPS macrodomains by microscopy and biochemistry approaches. We show for the first time that LPS macrodomains act as detergent resistant membranes (DRMs), segregating several lipid-raft components, LPS-binding proteins and MHC-II molecules. Brucella LPS macrodomains remain intact for several months in macrophages and are resistant to the disruptive effects of methyl β β β β -cyclodextrin. Fluorescent anisotropy measurements show that B. abortus LPS is responsible for the formation of rigid surface membrane complexes. In addition, relocalization of MHC-II molecules is observed in these structures. The effects of B. abortus LPS on membrane properties could be responsible for pathogenic effects such as the inhibition of MHC-II-dependent antigen presentation.