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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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Purified fusion factors mediate distinct steps in vesicle  docking and fusion. Saturating amounts of fusion factors (Sec18p  [50 ng], Uso1p [75 ng], LMA1 [50 ng]) and 2 ng/μl COPII proteins were mixed in various combinations with semi-intact cells.  35S-labeled gp-α-factor contained in freely diffusible vesicles (black  bars) and Golgi modified forms (hatched bars) were determined  after 45 min at 23°C. The incubation on the far right (+) contains  an additional 8 ng/μl of Sec23p complex.
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Figure 9: Purified fusion factors mediate distinct steps in vesicle docking and fusion. Saturating amounts of fusion factors (Sec18p [50 ng], Uso1p [75 ng], LMA1 [50 ng]) and 2 ng/μl COPII proteins were mixed in various combinations with semi-intact cells. 35S-labeled gp-α-factor contained in freely diffusible vesicles (black bars) and Golgi modified forms (hatched bars) were determined after 45 min at 23°C. The incubation on the far right (+) contains an additional 8 ng/μl of Sec23p complex.

Mentions: The initial cell-free assay for ER to Golgi transport (Baker et al., 1988) required semi-intact yeast cells and cytosol. If the vesicle fusion assay used in this current study represents an authentic subreaction of the overall transport process, these isolated fusion factors should be required for overall transport. Furthermore, if vesicle budding is a prerequisite for membrane fusion, the isolated fusion factors should be most effective in the presence of the COPII proteins (Sar1p, Sec23p complex, and Sec13p complex) that drive vesicle formation (i.e., fusion is coupled to budding). Washed semi-intact cells were first incubated with levels of COPII proteins that promote maximal budding efficiencies in the presence of purified fusion factors. Overall transport could not be detected above background under these initial conditions. However, a titration revealed that high concentrations of COPII proteins inhibit transport whereas lower concentrations of the coat constituents stimulate transport, even though suboptimal for vesicle budding (Fig. 8 A). Transport of 35S-labeled gp-α-factor to the Golgi was maximally 21% efficient in the reconstituted reaction compared to a background of 3%. As observed in Figs. 8 and 9, both COPII (2 ng/μl) and purified fusion factors were necessary for overall ER to Golgi transport in semi-intact cells indicating vesicle budding is a prerequisite for membrane fusion. To determine if the production of vesicle intermediates simply satisfies a spatial separation of the ER and Golgi present in semi-intact cells, a similar experiment was performed using diffusible ER (microsomes) and acceptor membranes incubated with fusion factors in the presence or absence of COPII (2 ng/μl) as in Fig. 8 A. Under this condition, addition of both fusion factors and COPII were again required for a maximal stimulation of transport (not shown), similar to that observed in semi-intact cells. These results suggest that COPII budding activates factor(s) involved in membrane fusion and that direct fusion of ER with Golgi membranes is not efficient.


Coupled ER to Golgi transport reconstituted with purified cytosolic proteins.

Barlowe C - J. Cell Biol. (1997)

Purified fusion factors mediate distinct steps in vesicle  docking and fusion. Saturating amounts of fusion factors (Sec18p  [50 ng], Uso1p [75 ng], LMA1 [50 ng]) and 2 ng/μl COPII proteins were mixed in various combinations with semi-intact cells.  35S-labeled gp-α-factor contained in freely diffusible vesicles (black  bars) and Golgi modified forms (hatched bars) were determined  after 45 min at 23°C. The incubation on the far right (+) contains  an additional 8 ng/μl of Sec23p complex.
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Related In: Results  -  Collection

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Figure 9: Purified fusion factors mediate distinct steps in vesicle docking and fusion. Saturating amounts of fusion factors (Sec18p [50 ng], Uso1p [75 ng], LMA1 [50 ng]) and 2 ng/μl COPII proteins were mixed in various combinations with semi-intact cells. 35S-labeled gp-α-factor contained in freely diffusible vesicles (black bars) and Golgi modified forms (hatched bars) were determined after 45 min at 23°C. The incubation on the far right (+) contains an additional 8 ng/μl of Sec23p complex.
Mentions: The initial cell-free assay for ER to Golgi transport (Baker et al., 1988) required semi-intact yeast cells and cytosol. If the vesicle fusion assay used in this current study represents an authentic subreaction of the overall transport process, these isolated fusion factors should be required for overall transport. Furthermore, if vesicle budding is a prerequisite for membrane fusion, the isolated fusion factors should be most effective in the presence of the COPII proteins (Sar1p, Sec23p complex, and Sec13p complex) that drive vesicle formation (i.e., fusion is coupled to budding). Washed semi-intact cells were first incubated with levels of COPII proteins that promote maximal budding efficiencies in the presence of purified fusion factors. Overall transport could not be detected above background under these initial conditions. However, a titration revealed that high concentrations of COPII proteins inhibit transport whereas lower concentrations of the coat constituents stimulate transport, even though suboptimal for vesicle budding (Fig. 8 A). Transport of 35S-labeled gp-α-factor to the Golgi was maximally 21% efficient in the reconstituted reaction compared to a background of 3%. As observed in Figs. 8 and 9, both COPII (2 ng/μl) and purified fusion factors were necessary for overall ER to Golgi transport in semi-intact cells indicating vesicle budding is a prerequisite for membrane fusion. To determine if the production of vesicle intermediates simply satisfies a spatial separation of the ER and Golgi present in semi-intact cells, a similar experiment was performed using diffusible ER (microsomes) and acceptor membranes incubated with fusion factors in the presence or absence of COPII (2 ng/μl) as in Fig. 8 A. Under this condition, addition of both fusion factors and COPII were again required for a maximal stimulation of transport (not shown), similar to that observed in semi-intact cells. These results suggest that COPII budding activates factor(s) involved in membrane fusion and that direct fusion of ER with Golgi membranes is not efficient.

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