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J Biol Chem. 2014 Oct 3;289(40):28040-53. doi: 10.1074/jbc.M114.593913. Epub 2014 Aug 19.

Vesicle associated membrane protein 8 (VAMP8)-mediated zymogen granule exocytosis is dependent on endosomal trafficking via the constitutive-like secretory pathway.

Author information

1
From the Department of Nutritional Sciences, University of Wisconsin, Madison, Wisconsin 53706.
2
Institute of Molecular and Cellular Biology, National University of Singapore, Singapore 138673.
3
Departments of Medicine and Physiology, University of Toronto, Ontario M5S 1A8, Canada, and.
4
Department of Neurosurgery, Georgia Institute of Technology, Atlanta, Georgia 30322.
5
From the Department of Nutritional Sciences, University of Wisconsin, Madison, Wisconsin 53706, groby@nutrisci.wisc.edu.

Erratum in

  • J Biol Chem. 2014 Dec 19;289(51):35264. Giasano, Herbert Y [Corrected to Gaisano, Herbert Y].

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.

KEYWORDS:

Endosome; Exocytosis; Intracellular Trafficking; Pancreas; SNARE Proteins

PMID:
25138214
PMCID:
PMC4183833
DOI:
10.1074/jbc.M114.593913
[Indexed for MEDLINE]
Free PMC Article

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