The membranes were separated by centrifugation, and membrane-bound (pellet, P) or unbound (soluble, S) material was analysed by autoradiography. formed proteasomes at their site of function. and radiolabelled in reticulocyte lysate in the presence of CMM (+CMM) or liposomes (+Lipos) or in the absence of membranes (?MM). The membranes were separated by centrifugation, and membrane-bound (pellet, P) or unbound (soluble, S) material was analysed by autoradiography. (B) POMP was removed from CMMs by washing the membranes at pH11 or with urea. (C) Binding of POMP to membranes occurred in a stoichiometric manner. POMP and luciferase were expressed in the presence of increasing amounts of CMMs. Radioactive material from the pellet and the soluble fraction was evaluated by phosphoimaging. A graph of the quantitative evaluation of pelleted material (POMP versus luciferase) is usually shown. Oxprenolol HCl The pixel density of the 1 l sample was set to 1 1. A representative experiment is shown. (D) The association of POMP with floated membranes is usually salt stable. Supernatants of each wash step were immunoblotted for POMP or calnexin. CMM, canine pancreatic microsomal membrane; POMP, proteasome maturation protein. Next, we investigated the stability of POMP association with ER membranes using floated microsomes. POMP remained stably attached to floated microsomes even at a salt concentration of 0.75 M KCl (Fig 3D), whereas other proteasomal subunits were stripped from microsomes at lower salt concentrations (data not shown), confirming the data. In summary, these experiments strongly imply a significant and stable conversation of POMP Rabbit Polyclonal to PSEN1 (phospho-Ser357) with microsomal membranes in the presence (+) or absence (?) of CMMs and POMP are shown. (E) Two-dimensional separation of immunoprecipitated (AbC8) 13S complexes (1C7, pro2, pro6, 3, 4 and POMP) from membrane floated material of pulse-labelled cells. (F) Two-dimensional separation of immunoprecipitated (6-Ab) 16S complexes (1C7, pro1, pro2, pro6, 3, 4, pro5, pro7 and POMP) from membrane floated material of pulse-labelled cells. Note the low methionine content of 4 and the abnormal migration behaviour of Oxprenolol HCl 5 due to the low isoelectric point. Ab, antibody; CMM, canine pancreatic microsomal membrane; ER, endoplasmic reticulum; IP, immunoprecipitation; PAGE, polyacrylamide gel electrophoresis; POMP, proteasome maturation protein. To assess whether POMP binds Oxprenolol HCl to -rings, we established an system for -ring formation and to circumvent the problem of constantly proceeding assembly of pre20S isolated from cells. The complexes formed were separated by sucrose gradient ultracentrifugation (supplementary Fig 2B,C online). Native gel analysis of fractions 5C7 showed a single band corresponding to the theoretical size of an -ring (supplementary Fig 2D online). Immunoprecipitates from fractions 5C7 with an 6-antibody showed a complex of 1C7 and POMP, indicating that POMP interacts with -rings (Fig 4C). In agreement with a recent report, PACs were dispensable for -ring assembly (Hirano were soluble in the absence of microsomes and pelleted together with membranes, whereas -subunits expressed alone had only a weak tendency to pellet (Fig 4D). Thus, we concluded that POMPC-rings exist in an ER-bound state. Further support for our hypothesis comes from studies that localize PAC1 (also Down syndrome critical region gene 2 (DSCR2)) to the cytoplasm as well as to the ER (Possik and fractionated. See the supplementary information online for further details. Supplementary information is available at online (http://www.emboreports.org). Supplementary Material supplementary Information Click here to view.(283K, pdf) Acknowledgments D. Ludwig and E. Brger are acknowledged for their excellent technical assistance. We thank J.J. Monaco for providing the AbC8 antibody, P.M. van Endert for the ERAP1mAb and S. Oxprenolol HCl Murata for the FlagCPAC2 construct. This work was supported by SFB740 from the Deutsche Forschungsgemeinschaft to E.K. and P.-M.K..