A heterotrimeric G protein, G alpha i-3, on Golgi membranes regulates the secretion of a heparan sulfate proteoglycan in LLC-PK1 epithelial cells

Abstract

A heterotrimeric G-alpha-i subunit, alpha-i-3, is localized on Golgi membranes in LLC-PK1 and NRK epithelial cells where it colocalizes with mannosidase II by immunofluorescence. The alpha-i-3 was found to be localized on the cytoplasmic face of Golgi cisternae and it was distributed across the whole Golgi stack. The alpha-i-3 subunit is found on isolated rat liver Golgi membranes by Western blotting and G-alpha-i-3 on the Golgi apparatus is ADP ribosylated by pertussis toxin. LLC-PK1 cells were stably transfected with G-alpha-i-3 on an MT-1, inducible promoter in order to overexpress alpha-i-3 on Golgi membranes. The intracellular processing and constitutive secretion of the basement membrane heparan sulfate proteoglycan (HSPG) was measured in LLC-PK1 cells. Overexpression of alpha-i-3 on Golgi membranes in transfected cells retarded the secretion of HSPG and accumulated precursors in the medial-trans-Golgi. This effect was reversed by treatment of cells with pertussis toxin which results in ADP-ribosylation and functional uncoupling of G-alpha-i-3 on Golgi membranes. These results provide evidence for a novel role for the pertussis toxin sensitive G-alpha-i-3 protein in Golgi trafficking of a constitutively secreted protein in epithelial cells