Role of clathrin in the regulated secretory pathway of pancreatic beta-cells

The role of clathrin in the sorting of proinsulin to secretory granules, th e formation of immature granules and their subsequent maturation is not kno wn. To this end, primary rat pancreatic beta -cells were infected with a re combinant adenovirus co-expressing the Hub fragment, a dominant-negative pe ptide of the clathrin heavy chain and enhanced green fluorescent protein (E GFP as a marker of infected cells). A population of cells expressing the hi ghest levels of EGFP (and thus Hub) was obtained using a fluorescence-activ ated cell sorter (FACS). Control cells were infected with an adenovirus exp ressing EGFP alone. By immunofluorescence, control cells showed intense sta ining for both clathrin light chain and proinsulin in a perinuclear region. In cells expressing high levels of Hub, the clathrin light-chain signal wa s faint and diffuse in keeping with its displacement from membranes. There was, however, no detectable effect of Hub expression on proinsulin staining or disposition within the cell. Proinsulin sorting and conversion, and the fate (release and/or degradation) of insulin and C-peptide, was studied by pulse-chase and quantitative reverse phase HPLC. In both Hub-expressing an d control cells, >99% of all newly synthesized proinsulin was sorted to the regulated pathway and there was no effect of Hub on proinsulin conversion to insulin. In presence of Hub there was, however, a significant increase i n the percentage of C-peptide truncated to des-(27-3l)-C-peptide at early t imes of chase as well as more extensive degradation of C-peptide thereafter . It is concluded that clathrin is not implicated in the sorting or process ing of proinsulin or in regulated exocytosis of secretory granules. These r esults confirm a role for clathrin in the removal of proteases from maturin g granules, thus explaining the increased truncation and degradation of C-p eptide in cells expressing Hub.