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Coupled ER to Golgi transport reconstituted with purified cytosolic proteins.

Barlowe C - J. Cell Biol. (1997)

Bottom Line: Manipulation of the semi-intact cell assay is used to distinguish freely diffusible ER- derived vesicles containing pro-alpha-factor from docked vesicles and from fused vesicles.Uso1p mediates vesicle docking and produces a dilution resistant intermediate.Ordering experiments using the dilution resistant intermediate and reversible Sec23p complex inhibition indicate Sec18p action is required before LMA1 function.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, Dartmouth Medical School, Hanover, New Hampshire 03755, USA. barlowe@dartmouth.edu

ABSTRACT
A cell-free vesicle fusion assay that reproduces a subreaction in transport of pro-alpha-factor from the ER to the Golgi complex has been used to fractionate yeast cytosol. Purified Sec18p, Uso1p, and LMA1 in the presence of ATP and GTP satisfies the requirement for cytosol in fusion of ER-derived vesicles with Golgi membranes. Although these purified factors are sufficient for vesicle docking and fusion, overall ER to Golgi transport in yeast semi-intact cells depends on COPII proteins (components of a membrane coat that drive vesicle budding from the ER). Thus, membrane fusion is coupled to vesicle formation in ER to Golgi transport even in the presence of saturating levels of purified fusion factors. Manipulation of the semi-intact cell assay is used to distinguish freely diffusible ER- derived vesicles containing pro-alpha-factor from docked vesicles and from fused vesicles. Uso1p mediates vesicle docking and produces a dilution resistant intermediate. Sec18p and LMA1 are not required for the docking phase, but are required for efficient fusion of ER- derived vesicles with the Golgi complex. Surprisingly, elevated levels of Sec23p complex (a subunit of the COPII coat) prevent vesicle fusion in a reversible manner, but do not interfere with vesicle docking. Ordering experiments using the dilution resistant intermediate and reversible Sec23p complex inhibition indicate Sec18p action is required before LMA1 function.

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Separation of fusion factors by Mono Q anion  exchange chromatography.  Isolated ER-derived vesicles  containing 35S-labeled gp- α-factor and acceptor membranes were incubated in a  30-μl reaction with the following protein fractions: 50  μg of cytosol (CYT), 50 μg of  the Q flowthrough (QFT), 50  μg of the 0.75 M eluate  (Q.75), or 15 μg of the 1.5 M  eluate (Q1.5). The percent  vesicle fusion represents the  amount of 35S-labeled gp-α-factor that has been modified by the addition of Golgi  specific α-1,6-mannose residues. In this experiment, the amount  of background fusion (complete reaction minus cytosol) was  2.7%.
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Figure 1: Separation of fusion factors by Mono Q anion exchange chromatography. Isolated ER-derived vesicles containing 35S-labeled gp- α-factor and acceptor membranes were incubated in a 30-μl reaction with the following protein fractions: 50 μg of cytosol (CYT), 50 μg of the Q flowthrough (QFT), 50 μg of the 0.75 M eluate (Q.75), or 15 μg of the 1.5 M eluate (Q1.5). The percent vesicle fusion represents the amount of 35S-labeled gp-α-factor that has been modified by the addition of Golgi specific α-1,6-mannose residues. In this experiment, the amount of background fusion (complete reaction minus cytosol) was 2.7%.

Mentions: Cytosol was bound to an anion exchange resin and three fractions were obtained: the flow through (QFT), a spectrum of proteins that elute at an intermediate ionic strength (Q.75), and proteins that elute at a high ionic strength (Q1.5). The individual fractions were dialyzed and tested in a cell-free assay for promotion of vesicle fusion (Fig. 1). Addition of individual fractions revealed that none of the fractions alone could drive vesicle fusion as efficiently as a crude cytosol, though a significant signal could be detected by adding saturating amounts of the Q1.5 fraction alone (Fig. 1, columns 1–4). A maximal signal (comparable to cytosol) could be obtained by combining all three of the fractions, and in fact, omission of either fraction resulted in a fusion efficiency below crude cytosol (Fig. 1, columns 5–8).


Coupled ER to Golgi transport reconstituted with purified cytosolic proteins.

Barlowe C - J. Cell Biol. (1997)

Separation of fusion factors by Mono Q anion  exchange chromatography.  Isolated ER-derived vesicles  containing 35S-labeled gp- α-factor and acceptor membranes were incubated in a  30-μl reaction with the following protein fractions: 50  μg of cytosol (CYT), 50 μg of  the Q flowthrough (QFT), 50  μg of the 0.75 M eluate  (Q.75), or 15 μg of the 1.5 M  eluate (Q1.5). The percent  vesicle fusion represents the  amount of 35S-labeled gp-α-factor that has been modified by the addition of Golgi  specific α-1,6-mannose residues. In this experiment, the amount  of background fusion (complete reaction minus cytosol) was  2.7%.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2140203&req=5

Figure 1: Separation of fusion factors by Mono Q anion exchange chromatography. Isolated ER-derived vesicles containing 35S-labeled gp- α-factor and acceptor membranes were incubated in a 30-μl reaction with the following protein fractions: 50 μg of cytosol (CYT), 50 μg of the Q flowthrough (QFT), 50 μg of the 0.75 M eluate (Q.75), or 15 μg of the 1.5 M eluate (Q1.5). The percent vesicle fusion represents the amount of 35S-labeled gp-α-factor that has been modified by the addition of Golgi specific α-1,6-mannose residues. In this experiment, the amount of background fusion (complete reaction minus cytosol) was 2.7%.
Mentions: Cytosol was bound to an anion exchange resin and three fractions were obtained: the flow through (QFT), a spectrum of proteins that elute at an intermediate ionic strength (Q.75), and proteins that elute at a high ionic strength (Q1.5). The individual fractions were dialyzed and tested in a cell-free assay for promotion of vesicle fusion (Fig. 1). Addition of individual fractions revealed that none of the fractions alone could drive vesicle fusion as efficiently as a crude cytosol, though a significant signal could be detected by adding saturating amounts of the Q1.5 fraction alone (Fig. 1, columns 1–4). A maximal signal (comparable to cytosol) could be obtained by combining all three of the fractions, and in fact, omission of either fraction resulted in a fusion efficiency below crude cytosol (Fig. 1, columns 5–8).

Bottom Line: Manipulation of the semi-intact cell assay is used to distinguish freely diffusible ER- derived vesicles containing pro-alpha-factor from docked vesicles and from fused vesicles.Uso1p mediates vesicle docking and produces a dilution resistant intermediate.Ordering experiments using the dilution resistant intermediate and reversible Sec23p complex inhibition indicate Sec18p action is required before LMA1 function.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, Dartmouth Medical School, Hanover, New Hampshire 03755, USA. barlowe@dartmouth.edu

ABSTRACT
A cell-free vesicle fusion assay that reproduces a subreaction in transport of pro-alpha-factor from the ER to the Golgi complex has been used to fractionate yeast cytosol. Purified Sec18p, Uso1p, and LMA1 in the presence of ATP and GTP satisfies the requirement for cytosol in fusion of ER-derived vesicles with Golgi membranes. Although these purified factors are sufficient for vesicle docking and fusion, overall ER to Golgi transport in yeast semi-intact cells depends on COPII proteins (components of a membrane coat that drive vesicle budding from the ER). Thus, membrane fusion is coupled to vesicle formation in ER to Golgi transport even in the presence of saturating levels of purified fusion factors. Manipulation of the semi-intact cell assay is used to distinguish freely diffusible ER- derived vesicles containing pro-alpha-factor from docked vesicles and from fused vesicles. Uso1p mediates vesicle docking and produces a dilution resistant intermediate. Sec18p and LMA1 are not required for the docking phase, but are required for efficient fusion of ER- derived vesicles with the Golgi complex. Surprisingly, elevated levels of Sec23p complex (a subunit of the COPII coat) prevent vesicle fusion in a reversible manner, but do not interfere with vesicle docking. Ordering experiments using the dilution resistant intermediate and reversible Sec23p complex inhibition indicate Sec18p action is required before LMA1 function.

Show MeSH
Related in: MedlinePlus