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Coupled ER to Golgi transport reconstituted with purified cytosolic proteins.

Barlowe C - J. Cell Biol. (1997)

Bottom Line: Uso1p mediates vesicle docking and produces a dilution resistant intermediate.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.

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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Purification of functional Uso1p-myc. Fractions eluting  from a Superose 6 column were analyzed by SDS-PAGE and silver stained (A), immunoblotted with anti-myc mAb (B), and  then assayed for stimulation of vesicle fusion in the presence of  QFT and Sec18p-6His (C). Fraction 17 contains the peak of activity and anti-myc immunoreactivity. Activity data represents vesicle  fusion above the QFT and Sec18p level that was 7% in this experiment.
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Figure 5: Purification of functional Uso1p-myc. Fractions eluting from a Superose 6 column were analyzed by SDS-PAGE and silver stained (A), immunoblotted with anti-myc mAb (B), and then assayed for stimulation of vesicle fusion in the presence of QFT and Sec18p-6His (C). Fraction 17 contains the peak of activity and anti-myc immunoreactivity. Activity data represents vesicle fusion above the QFT and Sec18p level that was 7% in this experiment.

Mentions: To confirm that Uso1p was the active component and to guide purification efforts, a version of Uso1p was constructed whereby the COOH terminus of the protein contains an additional 11 amino acid residues comprising a c-myc epitope recognized by mAb 9E10 (Evan et al., 1985). Expression of Uso1p-myc from a multicopy vector in a uso1-1 temperature sensitive strain fully complements when grown at 37°C indicating the c-myc extension does not interfere with Uso1p function. This strain was used to overproduce and purify Uso1p-myc by a two-step procedure using anion exchange and gel filtration chromatography as described under Materials and Methods. The final purification step (elution from the Superose 6 column) is shown in Fig. 5. The peak of fusion activity that elutes from the Superose 6 column at 800 kD coincided with a single 210-kD polypeptide species observed on SDS–polyacrylamide gel and c-myc immunoreactivity. These fractionation properties are consistent with previous studies suggesting Uso1p forms a nonglobular oligomer due to an extended coiled-coil rod structure similar to a related protein in mammalian cells, termed p115 (Waters et al., 1992; Seog et al., 1994; Sapperstein et al., 1995). The peak fractions eluting from the Superose 6 column were pooled (16–17) for use in later experiments, and neither Sec7p nor Sec26p (β-COP) could be detected in this pooled fraction by immunoblot analysis.


Coupled ER to Golgi transport reconstituted with purified cytosolic proteins.

Barlowe C - J. Cell Biol. (1997)

Purification of functional Uso1p-myc. Fractions eluting  from a Superose 6 column were analyzed by SDS-PAGE and silver stained (A), immunoblotted with anti-myc mAb (B), and  then assayed for stimulation of vesicle fusion in the presence of  QFT and Sec18p-6His (C). Fraction 17 contains the peak of activity and anti-myc immunoreactivity. Activity data represents vesicle  fusion above the QFT and Sec18p level that was 7% in this experiment.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 5: Purification of functional Uso1p-myc. Fractions eluting from a Superose 6 column were analyzed by SDS-PAGE and silver stained (A), immunoblotted with anti-myc mAb (B), and then assayed for stimulation of vesicle fusion in the presence of QFT and Sec18p-6His (C). Fraction 17 contains the peak of activity and anti-myc immunoreactivity. Activity data represents vesicle fusion above the QFT and Sec18p level that was 7% in this experiment.
Mentions: To confirm that Uso1p was the active component and to guide purification efforts, a version of Uso1p was constructed whereby the COOH terminus of the protein contains an additional 11 amino acid residues comprising a c-myc epitope recognized by mAb 9E10 (Evan et al., 1985). Expression of Uso1p-myc from a multicopy vector in a uso1-1 temperature sensitive strain fully complements when grown at 37°C indicating the c-myc extension does not interfere with Uso1p function. This strain was used to overproduce and purify Uso1p-myc by a two-step procedure using anion exchange and gel filtration chromatography as described under Materials and Methods. The final purification step (elution from the Superose 6 column) is shown in Fig. 5. The peak of fusion activity that elutes from the Superose 6 column at 800 kD coincided with a single 210-kD polypeptide species observed on SDS–polyacrylamide gel and c-myc immunoreactivity. These fractionation properties are consistent with previous studies suggesting Uso1p forms a nonglobular oligomer due to an extended coiled-coil rod structure similar to a related protein in mammalian cells, termed p115 (Waters et al., 1992; Seog et al., 1994; Sapperstein et al., 1995). The peak fractions eluting from the Superose 6 column were pooled (16–17) for use in later experiments, and neither Sec7p nor Sec26p (β-COP) could be detected in this pooled fraction by immunoblot analysis.

Bottom Line: Uso1p mediates vesicle docking and produces a dilution resistant intermediate.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.

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