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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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LMA1 replaces the  QFT fraction. In A, isolated vesicles and acceptor membranes  were incubated with Sec18p (50  ng) and Uso1p (75 ng) alone  (column 2) or with increasing  amounts of purified LMA1 (columns 3–6) in 30-μl reactions. In  B, immunoblots with antithioredoxin and anti-Sec18p are shown  to compare 10 ng of purified  LMA1 and Sec18p-6His with 5 μg  of crude cytosol (Load) and other  column fractions as described in  Fig. 2. The pure proteins loaded  represent ∼1/5 of the amount required for saturation and the  crude cytosol shown (Load) represents ∼1/10 the amount required for saturation.
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Figure 6: LMA1 replaces the QFT fraction. In A, isolated vesicles and acceptor membranes were incubated with Sec18p (50 ng) and Uso1p (75 ng) alone (column 2) or with increasing amounts of purified LMA1 (columns 3–6) in 30-μl reactions. In B, immunoblots with antithioredoxin and anti-Sec18p are shown to compare 10 ng of purified LMA1 and Sec18p-6His with 5 μg of crude cytosol (Load) and other column fractions as described in Fig. 2. The pure proteins loaded represent ∼1/5 of the amount required for saturation and the crude cytosol shown (Load) represents ∼1/10 the amount required for saturation.

Mentions: Sec18p/NSF is required for several intracellular transport steps and is considered a “general fusion factor” that uses ATP hydrolysis in fulfilling a role in membrane fusion (Rothman, 1994). Mutant sec18 yeast strains are defective for ER to Golgi transport in vivo and in vitro although this block has not been readily restored in various cell-free assays (Rexach and Schekman, 1991; Lupashin et al., 1996). Recombinant forms of NSF and Sec18p that contain six His residues at their COOH termini have been constructed, which may be overproduced in Escherichia coli (Whiteheart et al., 1994), and appear to be fully functional in cell-free assays (Whiteheart et al., 1994; Haas et al., 1996). Since Sec18p plays a central role in intracellular membrane fusion reactions, the six His-tagged protein was isolated in a buffer compatible with this assay and tested for promotion of vesicle fusion to the Golgi complex. Sec18p could completely substitute for the Q.75 fraction (Fig. 3 A) at a concentration similar to that contained in a saturating amount of this fraction (see Fig. 6 B). Furthermore, the purified Sec18p alone was not sufficient for fusion of ER-derived vesicles but required the addition of both the QFT and the Q1.5 for a maximal signal in the vesicle fusion assay (Fig. 3 A).


Coupled ER to Golgi transport reconstituted with purified cytosolic proteins.

Barlowe C - J. Cell Biol. (1997)

LMA1 replaces the  QFT fraction. In A, isolated vesicles and acceptor membranes  were incubated with Sec18p (50  ng) and Uso1p (75 ng) alone  (column 2) or with increasing  amounts of purified LMA1 (columns 3–6) in 30-μl reactions. In  B, immunoblots with antithioredoxin and anti-Sec18p are shown  to compare 10 ng of purified  LMA1 and Sec18p-6His with 5 μg  of crude cytosol (Load) and other  column fractions as described in  Fig. 2. The pure proteins loaded  represent ∼1/5 of the amount required for saturation and the  crude cytosol shown (Load) represents ∼1/10 the amount required for saturation.
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Related In: Results  -  Collection

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Figure 6: LMA1 replaces the QFT fraction. In A, isolated vesicles and acceptor membranes were incubated with Sec18p (50 ng) and Uso1p (75 ng) alone (column 2) or with increasing amounts of purified LMA1 (columns 3–6) in 30-μl reactions. In B, immunoblots with antithioredoxin and anti-Sec18p are shown to compare 10 ng of purified LMA1 and Sec18p-6His with 5 μg of crude cytosol (Load) and other column fractions as described in Fig. 2. The pure proteins loaded represent ∼1/5 of the amount required for saturation and the crude cytosol shown (Load) represents ∼1/10 the amount required for saturation.
Mentions: Sec18p/NSF is required for several intracellular transport steps and is considered a “general fusion factor” that uses ATP hydrolysis in fulfilling a role in membrane fusion (Rothman, 1994). Mutant sec18 yeast strains are defective for ER to Golgi transport in vivo and in vitro although this block has not been readily restored in various cell-free assays (Rexach and Schekman, 1991; Lupashin et al., 1996). Recombinant forms of NSF and Sec18p that contain six His residues at their COOH termini have been constructed, which may be overproduced in Escherichia coli (Whiteheart et al., 1994), and appear to be fully functional in cell-free assays (Whiteheart et al., 1994; Haas et al., 1996). Since Sec18p plays a central role in intracellular membrane fusion reactions, the six His-tagged protein was isolated in a buffer compatible with this assay and tested for promotion of vesicle fusion to the Golgi complex. Sec18p could completely substitute for the Q.75 fraction (Fig. 3 A) at a concentration similar to that contained in a saturating amount of this fraction (see Fig. 6 B). Furthermore, the purified Sec18p alone was not sufficient for fusion of ER-derived vesicles but required the addition of both the QFT and the Q1.5 for a maximal signal in the vesicle fusion assay (Fig. 3 A).

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