Technical Reference #1718

1718.

Organisation of human ER-exit sites: requirements for the localisation of Sec16 to transitional ER Helen Hughes1;*; Annika Budnik1;*; Katy Schmidt1;*; Krysten J. Palmer1; Judith Mantell1;3; Chris Noakes1;‡; Andrew Johnson1;§; Deborah A. Carter3; Paul Verkade1;2;3; Peter Watson1;¶ and David J. Stephens1;**, University of Bristol School of Medical Sciences, Journal of Cell Science, 122(1718), (2009)
Link To Paper

Abstract:
The COPII complex mediates the selective incorporation of secretory cargo and relevant machinery into budding vesicles at specialised sites on the endoplasmic reticulum membrane called transitional ER (tER). Here we show using confocal microscopy immunogold labelling of ultrathin cryosections and electron tomography that in human cells at steady state Sec16 localises to cup-like structures of tER that are spatially distinct from the localisation of other COPII coat components. We show that Sec16 defines the tER whereas Sec23-Sec24 and Sec13-Sec31 define later structures that precede but are distinct from the intermediate compartment. Steady-state localisation of Sec16 is independent of the localisation of downstream COPII components Sec23-Sec24 and Sec13-Sec31. Sec16 cycles on and off the membrane at a slower rate than other COPII components with a greater immobile fraction. We define the region of Sec16A that dictates its robust localisation of tER membranes and find that this requires both a highly charged region as well as a central domain that shows high sequence identity between species. The central conserved domain of Sec16 binds to Sec13 linking tER membrane localisation with COPII vesicle formation. These data are consistent with a model where Sec16 acts as a platform for COPII assembly at ERES.

Keywords:
COPII; Membrane traffic; secretion; vesicle formation

Materials & Methods:
Cells grown on live cell dishes (MatTek Ashland MA) were imaged at 37°C with the microscope LeicaTCS-SP5 AOBS scanning confocal microscope enclosed in a heated Perspex box in DMEM without phenol red supplemented with 30 mM HEPES pH 7.4 0.5 g/l sodium bicarbonate and 10% fetal calf serum. For quantitative FRAP measurements a 63 1.4 NA Plan-Apochromat objective was used. Photobleaching of GFP was performed with a ~500 nm diameter circular region. Pre- and post-bleach images were collected for 30 seconds using 15% AOTF power 20% laser intensity.

Microscopic Technique
Confocal Microscopy, Scanning

Cell Type(s)
HeLa