MUC2, the major colonic mucin, forms large polymers by N-terminal trimerization and C-terminal dimerization. Although the assembly process for MUC2 is established, it is not known how MUC2 is packed in the regulated secretory granulae of the goblet cell. When the N-terminal VWD1-D2-D′D3 domains (MUC2-N) were expressed in a goblet-like cell line, the protein was stored together with fulllength MUC2. By mimicking the pH and calcium conditions of the secretory pathway we analyzed purified MUC2-N by gel filtration, density gradient centrifugation, and transmission electron microscopy. At pH 7.4 the MUC2-N trimer eluted as a single peak by gel filtration. At pH 6.2 with Ca 2+ it formed large aggregates that did not enter the gel filtration column but were made visible after density gradient centrifugation. Electron microscopy studies revealed that the aggregates were composed of rings also observed in secretory granulae of colon tissue sections. The MUC2-N aggregates were dissolved by removing Ca 2+ and raising pH. After release from goblet cells, the unfolded full-length MUC2 formed stratified layers. These findings suggest a model for mucin packing in the granulae and the mechanism for mucin release, unfolding, and expansion.ucins are large glycoproteins that coat the surface of cells in the respiratory, digestive, and urogenital tracts (1, 2). Their main function is protection of epithelial cells from infection and physical injury. Mucins are characterized by mucin domains that are heavily O-glycosylated on the protein sequence rich in proline, threonine, and serine, therefore called PTS domains (3). These domains have little interspecies sequence conservation but often have tandemly repeated amino acid sequences that vary in number and length (3). There are several mucin types; the gelforming mucins are the only ones that form large polymers. In humans there are four gel-forming mucin genes that are known to be expressed, MUC2 in the intestine (4), MUC5AC in lungs and stomach, MUC5B in lungs and saliva, and MUC6 in stomach (1).MUC2 mucin is the major component of the mucus (mixture of mucins and other associated proteins) in the small and large intestine (2). In colon this is organized into two layers: an inner, densely packed layer that is attached to the epithelium that is impermeable to bacteria, and an outer, easily removable loose layer that is the habitat for the commensal bacteria (5). Human MUC2 mucin has 5,179 amino acids and contains multiple domains arranged in the following order (Fig. 1A): von Willebrand D1 domain (VWD1), VWD2, VWD′D3, (VWD1-D2-D′D3), first CysD, small PTS, second CysD, large PTS (tandemly repeated), C-terminal VWD4 followed by VWB, VWC, and a cystine-knot domain (CK) (4). The primary translational product of full-length MUC2 is quickly dimerized in the endoplasmic reticulum (ER) via disulfide bonds in the CK domain (6). The dimers pass into the Golgi apparatus, where the two PTS domains become O-glycosylated to form the two mucin domains. In the trans-Golgi network the glycosylated ...

Protein misfolding and aggregation is increasingly being recognized as a cause of disease. In Alzheimer’s disease the amyloid-β peptide (Aβ) misfolds into neurotoxic oligomers and assembles into amyloid fibrils. The Bri2 protein associated with Familial British and Danish dementias contains a BRICHOS domain, which reduces Aβ fibrillization as well as neurotoxicity in vitro and in a Drosophila model, but also rescues proteins from irreversible non-fibrillar aggregation. How these different activities are mediated is not known. Here we show that Bri2 BRICHOS monomers potently prevent neuronal network toxicity of Aβ, while dimers strongly suppress Aβ fibril formation. The dimers assemble into high-molecular-weight oligomers with an apparent two-fold symmetry, which are efficient inhibitors of non-fibrillar protein aggregation. These results indicate that Bri2 BRICHOS affects qualitatively different aspects of protein misfolding and toxicity via different quaternary structures, suggesting a means to generate molecular chaperone diversity.

electron microscopy ͉ transcription ͉ protein structure ͉ yeast ͉ Schizosaccharomyces pombe T he Mediator complex acts as an interface between genespecific regulatory proteins and the basal RNA polymerase II (pol II) transcription machinery (1). Mediator functions as a key regulator of pol II-dependent genes in Saccharomyces cerevisiae (2), and depletion of human Mediator from nuclear extracts abolishes transcription by pol II (3). The C-terminal domain of pol II (CTD) has an important role for the Mediator function (4, 5), and no fewer than nine SRB genes, encoding for Mediator subunits, were originally identified in a screen for mutants that suppress the cold-sensitive phenotype of a CTD truncation mutant (6). In S. cerevisiae and Schizosaccharomyces pombe, the Mediator complex interacts directly with the unphosphorylated CTD and forms a holoenzyme (5). Based on shape analysis of the low-resolution projection maps, the S. cerevisiae Mediator structure has been divided into three compact and visually distinguishable modules: head, middle, and tail domains of approximately equal mass (7).The subunit composition of S. cerevisiae Mediator has been studied in detail, and 21 proteins are bona fide members of the core Mediator complex (1,8,9). In addition, a subgroup of Srb proteins, Med12͞Srb8, Med13͞Srb9, Cdk8 (cyclin-dependent kinase 8)͞Srb10, and CycC͞Srb11, forms a specific module (the Cdk8 module) that is variably present in Mediator preparations (10, 11). The smaller, core Mediator (S Mediator) lacking the Cdk8 module has a stimulatory effect on basal transcription in vitro (5, 12). The larger form of Mediator (L Mediator), containing the Cdk8 module, instead represses basal transcription in vitro, and genetic analysis also indicates that the Cdk8 module is involved in the negative regulation of genes in vivo (13).The Cdk8 module influences pol II interactions with Mediator, and only S Mediator can interact with pol II and form a holoenzyme complex (11). The molecular mechanism by which the Cdk8 module negatively regulates pol II interactions has not been clarified, but it has been hypothesized that the negative effect of the Cdk8 module on eukaryotic transcription is caused by Cdk8-dependent phosphorylation of CTD. The hyperphosphorylated form of CTD would bind less tightly to Mediator, which may result in dissociation of pol II from the holoenzyme complex (14, 15).Here, we use the S. pombe system to investigate the molecular basis for the distinct functional properties of S and L Mediator. We find that the Cdk8 module binds to the pol II-binding cleft of Mediator, where it sterically blocks interactions with the polymerase. In contrast to earlier assumptions, the Cdk8 kinase activity is dispensable for negative regulation of pol II interactions with Mediator. It should be noted that the structure and function of Mediator appears conserved in fungi and metazoan cells, and to simplify comparisons with other experimental systems, throughout this study we use the recently proposed unifying Mediator nomenclature ...