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J. Biol. Chem., Vol. 280, Issue 17, 16868-16881, April 29, 2005
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From the Cystic Fibrosis/Pulmonary Research and Treatment Center, School of Medicine, University of North Carolina, Chapel Hill, North Carolina 27599-7248
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 (
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.
Received for publication, November 24, 2004 , and in revised form, January 31, 2005.
* This work was supported by Cystic Fibrosis Foundation Grants PEREZ3I0 and PEREZV04G0 (to J. P.-V.), National Institutes of Health Grant DK67404 (to J. P.-V.), and a grant from the University of North Carolina Research Council (to J. P.-V.). Part of this work was presented at the 2004 Annual Meeting of the Biophysical Society, February 1418, 2004, Baltimore. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed: Cystic Fibrosis/Pulmonary Research and Treatment Center, School of Medicine, CB7248, University of North Carolina, Chapel Hill, NC 27599-7248. Tel.: 919-843-5142; E-mail: juan_vilar{at}med.unc.edu.
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