pH-dependent intraluminal organization of mucin granules in live human mucous/goblet cells

被引:29
作者
Perez-Vilar, J [1 ]
Olsen, JC [1 ]
Chua, M [1 ]
Boucher, RC [1 ]
机构
[1] Univ N Carolina, Sch Med, Cyst Fibrosis Pulm Res & Treatment Ctr, Chapel Hill, NC 27599 USA
关键词
D O I
10.1074/jbc.M413289200
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
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 A(1), a specific inhibitor of the vacuolar H+-ATPase, did not alter the fusion protein mobility, although it significantly increased ( similar to 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.
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收藏
页码:16868 / 16881
页数:14
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