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

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.

[1]  J. Stow,et al.  Disruption of microtubules alters polarity of basement membrane proteoglycan secretion in epithelial cells. , 1991, The American journal of physiology.

[2]  L. Ercolani,et al.  Regulation of the G-protein alpha i-2 subunit gene in LLC-PK1 renal cells and isolation of porcine genomic clones encoding the gene promoter. , 1991, The Journal of biological chemistry.

[3]  H. Cantiello,et al.  G alpha i-3 regulates epithelial Na+ channels by activation of phospholipase A2 and lipoxygenase pathways. , 1990, The Journal of biological chemistry.

[4]  W. Huttner,et al.  Requirement for GTP hydrolysis in the formation of secretory vesicles , 1990, Nature.

[5]  L. Ercolani,et al.  Membrane localization of the pertussis toxin-sensitive G-protein subunits αi-2 and αi-3 and expression of a metallothionein-αi-2 fusion gene in LLC-PK1 cells , 1990, Proceedings of the National Academy of Sciences of the United States of America.

[6]  J. Rothman A molecular dissection of vesicular transport , 1990 .

[7]  M. Zerial,et al.  Localization of low molecular weight GTP binding proteins to exocytic and endocytic compartments , 1990, Cell.

[8]  B. Goud,et al.  Small GTP-binding protein associated with Golgi cisternae , 1990, Nature.

[9]  J. Rothman,et al.  Movement of proteins through the Golgi stack: a molecular dissection of vesicular transport 1 , 1990, FASEB journal : official publication of the Federation of American Societies for Experimental Biology.

[10]  D. Botstein,et al.  ADP-ribosylation factor is functionally and physically associated with the Golgi complex. , 1990, Proceedings of the National Academy of Sciences of the United States of America.

[11]  H. Cantiello,et al.  G protein subunit, alpha i-3, activates a pertussis toxin-sensitive Na+ channel from the epithelial cell line, A6. , 1989, The Journal of biological chemistry.

[12]  W. Simonds,et al.  Gi2 mediates alpha 2-adrenergic inhibition of adenylyl cyclase in platelet membranes: in situ identification with G alpha C-terminal antibodies. , 1989, Proceedings of the National Academy of Sciences of the United States of America.

[13]  G. Milligan,et al.  Distribution of G-proteins in rat liver plasma-membrane domains and endocytic pathways. , 1989, The Biochemical journal.

[14]  W. Balch,et al.  Calcium and GTP: essential components in vesicular trafficking between the endoplasmic reticulum and Golgi apparatus , 1989, The Journal of cell biology.

[15]  R. Ruddon,et al.  Biosynthesis and deposition of a noncovalent laminin-heparan sulfate proteoglycan complex and other basal lamina components by a human malignant cell line. , 1989, The Journal of biological chemistry.

[16]  G. Blobel,et al.  Identification of a G protein in rough endoplasmic reticulum of canine pancreas. , 1988, The Journal of biological chemistry.

[17]  S. Ferro-Novick,et al.  Reconstitution of protein transport from the endoplasmic reticulum to the Golgi complex in yeast: the acceptor Golgi compartment is defective in the sec23 mutant , 1988, The Journal of cell biology.

[18]  H. Bourne Do GTPases direct membrane traffic in secretion? , 1988, Cell.

[19]  N. Walworth,et al.  A GTP-binding protein required for secretion rapidly associates with secretory vesicles and the plasma membrane in yeast , 1988, Cell.

[20]  D. Botstein,et al.  The yeast GTP-binding YPT1 protein and a mammalian counterpart are associated with the secretion machinery , 1988, Cell.

[21]  J. Rothman,et al.  Involvement of GTP-binding “G” proteins in transport through the Golgi stack , 1987, Cell.

[22]  H. Anderson,et al.  Dependence on pH of polarized sorting of secreted proteins , 1987, Nature.

[23]  H. Cantiello,et al.  Inhibition of epithelial Na+ transport by atriopeptin, protein kinase c, and pertussis toxin. , 1987, The American journal of physiology.

[24]  P. Novick,et al.  A ras-like protein is required for a post-Golgi event in yeast secretion , 1987, Cell.

[25]  S. Cockcroft,et al.  The dual effector system for exocytosis in mast cells: Obligatory requirement for both Ca2+ and GTP , 1987, Bioscience reports.

[26]  K. Skorecki,et al.  Cross talk between stimulatory and inhibitory guanosine 5'-triphosphate binding proteins: role in activation and desensitization of the adenylate cyclase response to vasopressin. , 1987, Biochemistry.

[27]  J. Stow,et al.  Basement membrane heparan sulfate proteoglycans are concentrated in the laminae rarae and in podocytes of the rat renal glomerulus. , 1985, Proceedings of the National Academy of Sciences of the United States of America.

[28]  G. Warren,et al.  A monoclonal antibody against a 135‐K Golgi membrane protein. , 1982, The EMBO journal.

[29]  M. Farquhar,et al.  Sites of sulfate incorporation into mammotrophs and somatotrophs of the rat pituitary as determined by quantitative electron microscopic autoradiography. , 1980, Endocrinology.

[30]  K. Howell,et al.  Endoplasmic reticulum marker enzymes in Golgi fractions--what does this mean? , 1978, The Journal of cell biology.

[31]  George Palade,et al.  Intracellular Aspects of the Process of Protein Synthesis , 1975, Science.

[32]  P. Siekevitz,et al.  Golgi fractions prepared from rat liver homogenates. I. Isolation procedure and morphological characterization. , 1973 .

[33]  L. Ernster,et al.  AN ELECTRON-TRANSPORT SYSTEM ASSOCIATED WITH THE OUTER MEMBRANE OF LIVER MITOCHONDRIA , 1967, The Journal of cell biology.

[34]  A. Gilman,et al.  G proteins: transducers of receptor-generated signals. , 1987, Annual review of biochemistry.

[35]  L. Stryer,et al.  G proteins: a family of signal transducers. , 1986, Annual review of cell biology.

[36]  M. Farquhar Progress in unraveling pathways of Golgi traffic. , 1985, Annual review of cell biology.

[37]  L. Birnbaumer,et al.  ADP-ribosylation of membrane components by pertussis and cholera toxin. , 1985, Methods in enzymology.