title: pH-dependent intraluminal organization of mucin granules in live human mucous/goblet cells.
abstract: To study the mechanism of gel-forming mucin packaging within mucin granules, we generated human mucous/goblet cells stably expressing a recombinant MUC5AC domain fused to green fluorescent protein (GFP). The fusion protein, named SHGFP-MUC5AC/CK, accumulated in the granules together with native MUC5AC. Inhibition of protein synthesis or disorganization of the Golgi complex did not result in diminished intragranular SHGFP-MUC5AC/CK signals, consistent with long-term storage of the fusion protein. However, SHGFP-MUC5AC/CK was rapidly discharged from the granules upon incubation of the cells with ATP, an established mucin secretagogue. Several criteria indicated that SHGFP-MUC5AC/CK was not covalently linked to endogenous MUC5AC. Analysis of fluorescence recovery after photobleaching suggested that the intragranular SHGFP-MUC5AC/CK mobile fraction and mobility were significantly lower than in the endoplasmic reticulum lumen. Incubation of the cells with bafilomycin A1, a specific inhibitor of the vacuolar H+-ATPase, did not alter the fusion protein mobility, although it significantly increased (approximately 20%) the intragranular SHGFP-MUC5AC/CK mobile fraction. In addition, the granules in bafilomycin-incubated cells typically exhibited a heterogeneous intraluminal distribution of the fluorescent fusion protein. These results are consistent with a model of mucin granule intraluminal organization with two phases: a mobile phase in which secretory proteins diffuse as in the endoplasmic reticulum lumen but at a lower rate and an immobile phase or matrix in which proteins are immobilized by noncovalent pH-dependent interactions. An intraluminal acidic pH, maintained by the vacuolar H+-ATPase, is one of the critical factors for secretory protein binding to the immobile phase and also for its organization.
authors.affiliation: Cystic Fibrosis/Pulmonary Research and Treatment Center, School of Medicine, University of North Carolina, Chapel Hill, North Carolina 27599-7248, USA. juan_vilar@med.unc.edu
authors.forename: Juan
authors.initials: J
authors.lastname: Perez-Vilar
authors.forename: John C
authors.initials: JC
authors.lastname: Olsen
authors.forename: Michael
authors.initials: M
authors.lastname: Chua
authors.forename: Richard C
authors.initials: RC
authors.lastname: Boucher
mesh_terms: D000251:Adenosine Triphosphatases / Q000737:chemistry; D000255:Adenosine Triphosphate / Q000737:chemistry; D000595:Amino Acid Sequence; D000818:Animals; D019556:COS Cells; D002118:Calcium / Q000378:metabolism; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000378:metabolism; D004721:Endoplasmic Reticulum / Q000378:metabolism; D036681:Fluorescence Recovery After Photobleaching; D020397:Goblet Cells / Q000378:metabolism*; D049452:Green Fluorescent Proteins / Q000378:metabolism; D006801:Humans; D006863:Hydrogen-Ion Concentration; D018942:Macrolides / Q000494:pharmacology; D008854:Microscopy, Electron; D008969:Molecular Sequence Data; D055271:Mucin 5AC; D009077:Mucins / Q000737:chemistry* / Q000378:metabolism; D010641:Phenotype; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D015398:Signal Transduction; D013997:Time Factors
chemical_list: D018076:DNA, Complementary; C524678:MUC5AC protein, human; D018942:Macrolides; D055271:Mucin 5AC; D009077:Mucins; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; C057620:bafilomycin A; D049452:Green Fluorescent Proteins; C040929:bafilomycin A1; D000255:Adenosine Triphosphate; D000251:Adenosine Triphosphatases; D002118:Calcium