title: Proinsulin intermolecular interactions during secretory trafficking in pancreatic β cells.
abstract: Classically, exit from the endoplasmic reticulum (ER) is rate-limiting for secretory protein trafficking because protein folding/assembly occurs there. In this study, we have exploited "hPro-CpepSfGFP," a human proinsulin bearing "superfolder" green fluorescent C-peptide expressed in pancreatic β cells where it is processed to human insulin and CpepSfGFP. Remarkably, steady-state accumulation of hPro-CpepSfGFP and endogenous proinsulin is in the Golgi region, as if final stages of protein folding/assembly were occurring there. The Golgi regional distribution of proinsulin is dynamic, influenced by fasting/refeeding, and increased with β cell zinc deficiency. However, coexpression of ER-entrapped mutant proinsulin-C(A7)Y shifts the steady-state distribution of wild-type proinsulin to the ER. Endogenous proinsulin coprecipitates with hPro-CpepSfGFP and even more so with hProC(A7)Y-CpepSfGFP. Using Cerulean and Venus-tagged proinsulins, we find that both WT-WT and WT-mutant proinsulin pairs exhibit FRET. The data demonstrate that wild-type proinsulin dimerizes within the ER but accumulates at a poorly recognized slow step within the Golgi region, reflecting either slow kinetics of proinsulin hexamerization, steps in formation of nascent secretory granules, or other unknown molecular events. However, in the presence of ongoing misfolding of a subpopulation of proinsulin in β cells, the rate-limiting step in transport of the remaining proinsulin shifts to the ER.
authors.affiliation: Division of Metabolism, Endocrinology, and Diabetes, University of Michigan Medical School, Ann Arbor, Michigan 48105, USA.
authors.forename: Leena
authors.initials: L
authors.lastname: Haataja
authors.forename: Erik
authors.initials: E
authors.lastname: Snapp
authors.forename: Jordan
authors.initials: J
authors.lastname: Wright
authors.forename: Ming
authors.initials: M
authors.lastname: Liu
authors.forename: Alexandre B
authors.initials: AB
authors.lastname: Hardy
authors.forename: Michael B
authors.initials: MB
authors.lastname: Wheeler
authors.forename: Michele L
authors.initials: ML
authors.lastname: Markwardt
authors.forename: Megan A
authors.initials: MA
authors.lastname: Rizzo
authors.forename: Peter
authors.initials: P
authors.lastname: Arvan
mesh_terms: D000818:Animals; D002096:C-Peptide / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D019556:COS Cells; D045744:Cell Line, Tumor; D002522:Chlorocebus aethiops; D019281:Dimerization; D004721:Endoplasmic Reticulum / Q000235:genetics / Q000378:metabolism* / Q000648:ultrastructure; D015870:Gene Expression; D006056:Golgi Apparatus / Q000235:genetics / Q000378:metabolism* / Q000648:ultrastructure; D049452:Green Fluorescent Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D006801:Humans; D007328:Insulin / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D050417:Insulin-Secreting Cells / Q000166:cytology / Q000378:metabolism*; D007700:Kinetics; D051379:Mice; D018613:Microscopy, Confocal; D010957:Plasmids; D011485:Protein Binding; D017510:Protein Folding; D021381:Protein Transport; D051381:Rats; D014162:Transfection
chemical_list: D002096:C-Peptide; D007328:Insulin; D049452:Green Fluorescent Proteins