Monensin and brefeldin A differentially affect the phosphorylation and sulfation of secretory proteins.
Chromogranin B and secretogranin II, two members of the granin family, are known to be post-translationally modified by the addition of O-linked carbohydrates to serine and/or threonine, phosphate to serine and threonine, and sulfate to carbohydrate and tyrosine residues. In the present study, chromogranin B and secretogranin II were used as model proteins to investigate in which subcompartment of the Golgi complex secretory proteins become phosphorylated. Monensin, a drug known to block the transport from the medial to the trans cisternae of the Golgi stack, inhibited the phosphorylation of the granins, indicating that this modification occurred distal to the medial Golgi. Monensin also blocked the addition of galactose to O-linked carbohydrates and the sulfation of the granins, confirming previous data that these modifications take place in the trans Golgi. To distinguish, within the trans Golgi, between the trans cisternae of the Golgi stack and the trans Golgi network, we made use of the previous observation that brefeldin A results in the redistribution to the endoplasmic reticulum of membrane-bound enzymes of the trans cisternae of the Golgi stack, but not of the trans Golgi network. Brefeldin A treatment abolished granin sulfation but resulted in the accumulation of phosphorylated and galactosylated granins. Differential effects of brefeldin A on membranes of the Golgi stack versus the trans Golgi network were also observed by immunofluorescence analysis of marker proteins specific for either compartment. Our results suggest that the phosphorylation of secretory proteins, like their galactosylation, largely occurs in the trans cisternae of the Golgi stack, whereas the sulfation of secretory proteins on both carbohydrate and tyrosine residues takes place selectively in the trans Golgi network.
Rosa
P
P
Consiglio Nazionale delle Ricerche-Center of Cytopharmacology, Department of Pharmacology, Milan, Italy.
Mantovani
S
S
Rosboch
R
R
Huttner
W B
WB
D000818:Animals; D001345:Autoradiography; D020126:Brefeldin A; D002864:Chromogranins / Q000378:metabolism*; D003517:Cyclopentanes / Q000494:pharmacology*; D015180:Electrophoresis, Gel, Two-Dimensional; D004721:Endoplasmic Reticulum / Q000378:metabolism; D006056:Golgi Apparatus / Q000378:metabolism; D008985:Monensin / Q000494:pharmacology*; D016716:PC12 Cells; D010766:Phosphorylation / Q000187:drug effects; D011499:Protein Processing, Post-Translational; D011506:Proteins / Q000378:metabolism*; D051381:Rats; D013464:Sulfuric Acids / Q000378:metabolism*
D002864:Chromogranins; D003517:Cyclopentanes; D011506:Proteins; D013464:Sulfuric Acids; D020126:Brefeldin A; D008985:Monensin