The differential effects of dithiothreitol and 2-mercaptoethanol on the secretion of partially and completely assembled immunoglobulins suggest that thiol-mediated retention does not take place in or beyond the Golgi.
Dithiothreitol (DTT) blocks the endoplasmic reticulum (ER)-Golgi transport of newly synthesized immunoglobulin (Ig) molecules, whereas 2-mercaptoethanol (2ME) allows secretion of unpolymerized Igs otherwise retained intracellularly by disulphide interchange reactions. To understand this dichotomy, we have compared the effects of DTT and 2ME on the assembly, intracellular transport, and secretion of a panel of chimeric Igs that are either constitutively secreted or retained intracellularly. Our results demonstrate that DTT, but not 2ME, reduces some of the inter- and intrachain disulphide bonds and causes partial disassembly of H2L2 complexes and unfolding of individual chains in the ER. Upon DTT removal, heavy (H) and light (L) chains reform hapten-binding H2L2 molecules, which are later secreted. Reduction of the H2L2 interchain disulphide bonds can occur along the entire secretory pathway; however, in or beyond the Golgi this does not result in efficient H-L disassembly or unfolding. As a consequence, DTT does not block the exit from the Golgi. Moreover, unpolymerized Igs--normally retained in a pre-Golgi compartment--no longer require reducing agents to be secreted once they have reached the Golgi. Thus, little if any thiol-mediated retention seems to take place in or beyond the Golgi complex.
Valetti
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DIBIT-San Raffaele Scientific Institute, Milano, Italy.
Sitia
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D001692:Biological Transport / Q000187:drug effects; D002460:Cell Line; D004220:Disulfides / Q000378:metabolism; D004229:Dithiothreitol / Q000494:pharmacology*; D004721:Endoplasmic Reticulum / Q000378:metabolism; D005690:Galactose; D006056:Golgi Apparatus / Q000378:metabolism*; D007074:Immunoglobulin G / Q000096:biosynthesis / Q000187:drug effects*; D007075:Immunoglobulin M / Q000096:biosynthesis / Q000187:drug effects*; D007146:Immunoglobulin lambda-Chains / Q000096:biosynthesis / Q000187:drug effects; D008623:Mercaptoethanol / Q000494:pharmacology*; D017510:Protein Folding; D011499:Protein Processing, Post-Translational; D011993:Recombinant Fusion Proteins / Q000187:drug effects / Q000378:metabolism
D004220:Disulfides; D007074:Immunoglobulin G; D007075:Immunoglobulin M; D007146:Immunoglobulin lambda-Chains; D011993:Recombinant Fusion Proteins; D008623:Mercaptoethanol; D004229:Dithiothreitol; D005690:Galactose