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| TR |
TITLE |
AUTHORS |
KEYWORDS |
MATERIALS & METHODS
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MICROSCOPIC
TECHNIQUES |
SPECIES |
MORPHOLOGY |
CELL
LINE |
| 614 |
GBF1,
a cis-Golgi and VTCs-localized ARF-GEF, is
implicated in ER-to-Golgi protein traffic |
Xinhua
Zhao, Alejandro Claude, Justin Chun, David J. Shields, John F. Presley and Paul Melançon, |
GBF1,
ERGIC, VTCs, ARF-GEF, Brefeldin A, Protein
traffic |
For Fig. 7, NRK-GFP-GBF1 cells
were grown on Plastek Cultureware glass bottom
microwell dishes (MatTek Corp) and imaged on the temperature-controlled stage of a Zeiss Axiovert
200M microscope equipped with an UltraVIEW ERS 3E spinning disk.
|
confocal
microscopy |
|
|
NRK-GFP-GBF1 |
| Abstract |
| |
The formation and maturation of membrane carriers
that transport cargo from the ER to the Golgi complex involves the sequential action of the
coat protein complexes COPII and COPI. Recruitment of COPI to nascent carriers requires activation
of ADP-ribosylation factors by a BrefeldinA-sensitive guanine nucleotide exchange factor. Using
new antisera and a GFP-tagged protein, we demonstrate that the exchange factor GBF1 localized
to both Golgi membranes and peripheral puncta, near but separate from ER exit sites. Live cell
imaging revealed that GFP-GBF1 associates dynamically with both membranes through rapid exchange
with a large cytosolic pool. Treatment with BrefeldinA dramatically altered this rapid exchange,
causing accumulation of GBF1 on both Golgi and peripheral puncta before eventual redistribution
to the ER in a microtubule-dependent manner. Measurement of diffusion coefficients and subcellular
fractionation confirmed this shift in GBF1 from cytosolic to membrane bound. BrefeldinA-induced
accumulation of GBF1 coincided with loss of COPI from peripheral puncta. Furthermore, recruitment
of GBF1 to cargo-containing peripheral puncta coincided with recruitment of COPI, but not COPII.
Strikingly, microinjection of anti-GBF1 antibodies specifically caused dissociation of COPI
from membranes. These observations strongly suggest that GBF1 regulates COPI membrane recruitment
in the early secretory pathway. |
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