title: Vesicle associated membrane protein 8 (VAMP8)-mediated zymogen granule exocytosis is dependent on endosomal trafficking via the constitutive-like secretory pathway.
abstract: Acinar cell zymogen granules (ZG) express 2 isoforms of the vesicle-associated membrane protein family (VAMP2 and -8) thought to regulate exocytosis. Expression of tetanus toxin to cleave VAMP2 in VAMP8 knock-out (-/-) acini confirmed that VAMP2 and -8 are the primary VAMPs for regulated exocytosis, each contributing ∼50% of the response. Analysis of VAMP8(-/-) acini indicated that although stimulated secretion was significantly reduced, a compensatory increase in constitutive secretion maintained total secretion equivalent to wild type (WT). Using a perifusion system to follow secretion over time revealed VAMP2 mediates an early rapid phase peaking and falling within 2-3 min, whereas VAMP8 controls a second prolonged phase that peaks at 4 min and slowly declines over 20 min to support the protracted secretory response. VAMP8(-/-) acini show increased expression of the endosomal proteins Ti-VAMP7 (2-fold) and Rab11a (4-fold) and their redistribution from endosomes to ZGs. Expression of GDP-trapped Rab11a-S25N inhibited secretion exclusively from the VAMP8 but not the VAMP2 pathway. VAMP8(-/-) acini also showed a >90% decrease in the early endosomal proteins Rab5/D52/EEA1, which control anterograde trafficking in the constitutive-like secretory pathway. In WT acini, short term (14-16 h) culture also results in a >90% decrease in Rab5/D52/EEA1 and a complete loss of the VAMP8 pathway, whereas VAMP2-secretion remains intact. Remarkably, rescue of Rab5/D52/EEA1 expression restored the VAMP8 pathway. Expressed D52 shows extensive colocalization with Rab11a and VAMP8 and partially copurifies with ZG fractions. These results indicate that robust trafficking within the constitutive-like secretory pathway is required for VAMP8- but not VAMP2-mediated ZG exocytosis.
authors.affiliation: From the Department of Nutritional Sciences, University of Wisconsin, Madison, Wisconsin 53706.
authors.forename: Scott W
authors.initials: SW
authors.lastname: Messenger
authors.affiliation: From the Department of Nutritional Sciences, University of Wisconsin, Madison, Wisconsin 53706.
authors.forename: Michelle A
authors.initials: MA
authors.lastname: Falkowski
authors.affiliation: From the Department of Nutritional Sciences, University of Wisconsin, Madison, Wisconsin 53706.
authors.forename: Diana D H
authors.initials: DD
authors.lastname: Thomas
authors.affiliation: From the Department of Nutritional Sciences, University of Wisconsin, Madison, Wisconsin 53706.
authors.forename: Elaina K
authors.initials: EK
authors.lastname: Jones
authors.affiliation: Institute of Molecular and Cellular Biology, National University of Singapore, Singapore 138673.
authors.forename: Wanjin
authors.initials: W
authors.lastname: Hong
authors.forename: Herbert Y
authors.initials: HY
authors.lastname: Gaisano
authors.affiliation: Departments of Medicine and Physiology, University of Toronto, Ontario M5S 1A8, Canada, and.
authors.forename: Herbert Y
authors.initials: HY
authors.lastname: Giasano
authors.affiliation: Department of Neurosurgery, Georgia Institute of Technology, Atlanta, Georgia 30322.
authors.forename: Nicholas M
authors.initials: NM
authors.lastname: Boulis
authors.affiliation: From the Department of Nutritional Sciences, University of Wisconsin, Madison, Wisconsin 53706, groby@nutrisci.wisc.edu.
authors.forename: Guy E
authors.initials: GE
authors.lastname: Groblewski
mesh_terms: D061354:Acinar Cells / Q000378:metabolism; D000818:Animals; D011992:Endosomes / Q000235:genetics / Q000378:metabolism*; D005089:Exocytosis; D008297:Male; D008810:Mice, Inbred C57BL; D018345:Mice, Knockout; D021381:Protein Transport; D050683:R-SNARE Proteins / Q000235:genetics / Q000378:metabolism*; D055571:Secretory Pathway; D022142:Secretory Vesicles / Q000235:genetics / Q000378:metabolism*; D050988:Vesicle-Associated Membrane Protein 2 / Q000235:genetics / Q000378:metabolism
chemical_list: D050683:R-SNARE Proteins; C491831:Vamp8 protein, mouse; D050988:Vesicle-Associated Membrane Protein 2; C494808:vesicle-associated membrane protein 2, mouse
keywords: Endosome; Exocytosis; Intracellular Trafficking; Pancreas; SNARE Proteins