Activation of the transcription factor nuclear factor kappa B (NF-kappaB) is controlled by sequential phosphorylation, ubiquitination, and degradation of its inhibitory subunit IkappaB. A large multiprotein complex, the IkappaB kinase (IKK) signalsome, was purified from HeLa cells and found to contain a cytokine-inducible IkappaB kinase activity that phosphorylates IkappaB-alpha and IkappaB-beta. Two components of the IKK signalsome, IKK-1 and IKK-2, were identified as closely related protein serine kinases containing leucine zipper and helix-loop-helix protein interaction motifs. Mutant versions of IKK-2 had pronounced effects on RelA nuclear translocation and NF-kappaB-dependent reporter activity, consistent with a critical role for the IKK kinases in the NF-kappaB signaling pathway.

Activation of the transcription factor NF-B is controlled by the sequential phosphorylation, ubiquitination, and degradation of its inhibitory subunit, IB. We recently purified a large multiprotein complex, the IB kinase (IKK) signalsome, which contains two regulated IB kinases, IKK1 and IKK2, that can each phosphorylate IB␣ and IB␤. The IKK signalsome contains several additional proteins presumably required for the regulation of the NFB signal transduction cascade in vivo. In this report, we demonstrate reconstitution of IB kinase activity in vitro by using purified recombinant IKK1 and IKK2. Recombinant IKK1 or IKK2 forms homo-or heterodimers, suggesting the possibility that similar IKK complexes exist in vivo. Indeed, in HeLa cells we identified two distinct IKK complexes, one containing IKK1-IKK2 heterodimers and the other containing IKK2 homodimers, which display differing levels of activation following tumor necrosis factor alpha stimulation. To better elucidate the nature of the IKK signalsome, we set out to identify IKK-associated proteins. To this end, we purified and cloned a novel component common to both complexes, named IKKassociated protein 1 (IKKAP1). In vitro, IKKAP1 associated specifically with IKK2 but not IKK1.

Abstract. Using a novel in vitro assay which allows us to distinguish vesicle budding from subsequent targeting and fusion steps, we provide the first biological evidence that B-COP, a component of non-clathrincoated vesicles believed to mediate intraGolgi transport, is essential for transport of protein from the ER to the cis-Golgi compartment. Incubation in the presence of B-COP specific antibodies and Fab fragments prevents the exit of vesicular stomatitis virus glycoprotein (VSV-G) from the ER. These results demonstrate that fl-COP is required for the assembly of coat complexes mediating vesicle budding. Fractionation of rat liver cytosol revealed that a major biologically active form of B-COP was found in a high molecular pool (>1,000 kD) distinct from coatomer and which promoted efficient vesicle budding from the ER. Surprisingly, rablB could be quantitatively coprecipitated with this/3-COP containing complex and was also essential for function. We suggest that/3-COP functions in an early step during vesicle formation and that rablB may be recruited as a component of a precoat complex which participates in the export of protein from the ER via vesicular carders.

Leader peptidase (Lep) is a central component of the secretory machinery of Escherichia coli, where it serves to remove signal peptides from secretory proteins. It spans the inner membrane twice with a large C‐terminal domain protruding into the periplasmic space. To investigate the importance of the different structural domains for the catalytic activity, we have studied the effects of a large panel of Lep mutants on the processing of signal peptides, both in vivo and in vitro. Our data suggest that the first transmembrane and cytoplasmic regions are not directly involved in catalysis, but that the second transmembrane region and the region immediately following it may be in contact with the signal peptide and/or located spatially close to the active site of Lep.