Targeted siRNA Screen to Identify SNAREs Required for Constitutive Secretion in Mammalian Cells

The role of SNAREs in mammalian constitutive secretion remains poorly defined. To address this, we have developed a novel flow cytometry-based assay for measuring constitutive secretion and have performed a targeted SNARE and Sec1/Munc18 (SM) protein-specific siRNA screen (38 SNAREs, 4 SNARE-like pr...

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Bibliographic Details
Published in:Traffic (Copenhagen, Denmark) Denmark), 2010-09, Vol.11 (9), p.1191-1204
Main Authors: Gordon, David E, Bond, Lisa M, Sahlender, Daniela A, Peden, Andrew A
Format: Article
Language:English
Subjects:
Animals
assay
assays
CEDNIK
constitutive secretion
Flow Cytometry - methods
Humans
Protein Transport
Qa-SNARE Proteins - metabolism
Reverse Transcriptase Polymerase Chain Reaction
RNA, Small Interfering - genetics
Signal Transduction
SNAP-29
SNARE
SNARE Proteins - genetics
SNARE Proteins - metabolism
syntaxin
syntaxin 19
TIRF
VAMP
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Summary:The role of SNAREs in mammalian constitutive secretion remains poorly defined. To address this, we have developed a novel flow cytometry-based assay for measuring constitutive secretion and have performed a targeted SNARE and Sec1/Munc18 (SM) protein-specific siRNA screen (38 SNAREs, 4 SNARE-like proteins and 7 SM proteins). We have identified the endoplasmic reticulum (ER)/Golgi SNAREs syntaxin 5, syntaxin 17, syntaxin 18, GS27, SLT1, Sec20, Sec22b, Ykt6 and the SM protein Sly1, along with the post-Golgi SNAREs SNAP-29 and syntaxin 19, as being required for constitutive secretion. Depletion of SNAP-29 or syntaxin 19 causes a decrease in the number of fusion events at the cell surface and in SNAP-29-depleted cells causes an increase in the number of docked vesicles at the plasma membrane as determined by total internal reflection fluorescence (TIRF) microscopy. Analysis of syntaxin 19-interacting partners by mass spectrometry indicates that syntaxin 19 can form SNARE complexes with SNAP-23, SNAP-25, SNAP-29, VAMP3 and VAMP8, supporting its role in Golgi to plasma membrane transport or fusion. Surprisingly, we have failed to detect any requirement for a post-Golgi-specific R-SNARE in this process.
ISSN:1398-9219
1600-0854
DOI:10.1111/j.1600-0854.2010.01087.x