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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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Uso1p action generates a dilution resistant intermediate. Isolated vesicles were mixed on ice with acceptor membranes  and individual fusion factors Uso1p (U), LMA1 (L), and Sec18p  (18), or the set of fusion factors together (U/L/18). In these experiments, the concentrations of acceptor membranes, vesicles,  and fusion factors were half of the standard condition (described  in Fig. 7). After incubation at 23°C for various times, reactions  were diluted 10-fold with one of the following: buffer containing  ATP, LMA1, and Sec18p (L/18); buffer containing ATP, Uso1p,  and Sec18p (U/18); buffer containing ATP, Uso1p, and LMA1  (U/L); or buffer containing ATP alone (B88). Diluents contained  purified proteins at levels that produce initial concentrations of  each indicated species. Each tube was incubated at the reaction  temperature for a total of 90 min. In this experiment, background  fusion (vesicles, acceptor membranes, and ATP, undiluted) was  1.7% and maximal fusion (vesicles, acceptor membranes, fusion  factors, and ATP, undiluted) was 12%.
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Figure 10: Uso1p action generates a dilution resistant intermediate. Isolated vesicles were mixed on ice with acceptor membranes and individual fusion factors Uso1p (U), LMA1 (L), and Sec18p (18), or the set of fusion factors together (U/L/18). In these experiments, the concentrations of acceptor membranes, vesicles, and fusion factors were half of the standard condition (described in Fig. 7). After incubation at 23°C for various times, reactions were diluted 10-fold with one of the following: buffer containing ATP, LMA1, and Sec18p (L/18); buffer containing ATP, Uso1p, and Sec18p (U/18); buffer containing ATP, Uso1p, and LMA1 (U/L); or buffer containing ATP alone (B88). Diluents contained purified proteins at levels that produce initial concentrations of each indicated species. Each tube was incubated at the reaction temperature for a total of 90 min. In this experiment, background fusion (vesicles, acceptor membranes, and ATP, undiluted) was 1.7% and maximal fusion (vesicles, acceptor membranes, fusion factors, and ATP, undiluted) was 12%.

Mentions: Ordering of the Uso1p, Sec18p and LMA1 requirements was pursued in the reconstituted vesicle fusion assay because the semi-intact cell assay does not show a strict dependence on exogenously added Sec18p. If Uso1p tethers ER-derived vesicle to the Golgi compartment, the action of Uso1p may produce a dilution resistant intermediate. Indeed, results shown in Fig. 10 support this concept. In this experiment, the concentrations of vesicles, acceptor membranes and purified proteins were lowered (see Fig. 10, figure legend) to manipulate this intermediate. The maximal fusion efficiency was reduced under these conditions but a clear sensitivity to 10-fold dilution was observed (Fig. 10, •). Preincubation of vesicles and acceptor membranes with Uso1p alone generated a dilution resistant species that may be chased upon addition of Sec18p and LMA1 (Fig. 10, ▪). In contrast, incubation with Sec18p or LMA1 alone does not produce dilution resistance even when adequate levels of the missing fusion factors are supplied in the diluent. These results again suggest Uso1p function is independent from and precedes Sec18p and LMA1 action. Incubation with Uso1p alone followed by dilution with Sec18p and LMA1 (Fig. 10, ▪) was not the same as that observed for the complete reaction diluted with buffer containing ATP (Fig. 10, •). This was due to dilution of the Sec18p and LMA1 proteins under the latter condition whereas active concentrations of these fusion factors were maintained under the former. Therefore at early time points, Uso1p-docked vesicles efficiently chase due to maintenance of LMA1 and Sec18p throughout the second incubation but docked vesicles for the complete reaction (Fig. 10, •) were not chased as efficiently because of dilution of Sec18p and LMA1. At later times (20 and 30 min), the fusion efficiency of the Uso1p-docked intermediate was not as efficient as that observed with the complete reaction. This may be because of lability of the docked intermediate in the absence of Sec18p and LMA1.


Coupled ER to Golgi transport reconstituted with purified cytosolic proteins.

Barlowe C - J. Cell Biol. (1997)

Uso1p action generates a dilution resistant intermediate. Isolated vesicles were mixed on ice with acceptor membranes  and individual fusion factors Uso1p (U), LMA1 (L), and Sec18p  (18), or the set of fusion factors together (U/L/18). In these experiments, the concentrations of acceptor membranes, vesicles,  and fusion factors were half of the standard condition (described  in Fig. 7). After incubation at 23°C for various times, reactions  were diluted 10-fold with one of the following: buffer containing  ATP, LMA1, and Sec18p (L/18); buffer containing ATP, Uso1p,  and Sec18p (U/18); buffer containing ATP, Uso1p, and LMA1  (U/L); or buffer containing ATP alone (B88). Diluents contained  purified proteins at levels that produce initial concentrations of  each indicated species. Each tube was incubated at the reaction  temperature for a total of 90 min. In this experiment, background  fusion (vesicles, acceptor membranes, and ATP, undiluted) was  1.7% and maximal fusion (vesicles, acceptor membranes, fusion  factors, and ATP, undiluted) was 12%.
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Related In: Results  -  Collection

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Figure 10: Uso1p action generates a dilution resistant intermediate. Isolated vesicles were mixed on ice with acceptor membranes and individual fusion factors Uso1p (U), LMA1 (L), and Sec18p (18), or the set of fusion factors together (U/L/18). In these experiments, the concentrations of acceptor membranes, vesicles, and fusion factors were half of the standard condition (described in Fig. 7). After incubation at 23°C for various times, reactions were diluted 10-fold with one of the following: buffer containing ATP, LMA1, and Sec18p (L/18); buffer containing ATP, Uso1p, and Sec18p (U/18); buffer containing ATP, Uso1p, and LMA1 (U/L); or buffer containing ATP alone (B88). Diluents contained purified proteins at levels that produce initial concentrations of each indicated species. Each tube was incubated at the reaction temperature for a total of 90 min. In this experiment, background fusion (vesicles, acceptor membranes, and ATP, undiluted) was 1.7% and maximal fusion (vesicles, acceptor membranes, fusion factors, and ATP, undiluted) was 12%.
Mentions: Ordering of the Uso1p, Sec18p and LMA1 requirements was pursued in the reconstituted vesicle fusion assay because the semi-intact cell assay does not show a strict dependence on exogenously added Sec18p. If Uso1p tethers ER-derived vesicle to the Golgi compartment, the action of Uso1p may produce a dilution resistant intermediate. Indeed, results shown in Fig. 10 support this concept. In this experiment, the concentrations of vesicles, acceptor membranes and purified proteins were lowered (see Fig. 10, figure legend) to manipulate this intermediate. The maximal fusion efficiency was reduced under these conditions but a clear sensitivity to 10-fold dilution was observed (Fig. 10, •). Preincubation of vesicles and acceptor membranes with Uso1p alone generated a dilution resistant species that may be chased upon addition of Sec18p and LMA1 (Fig. 10, ▪). In contrast, incubation with Sec18p or LMA1 alone does not produce dilution resistance even when adequate levels of the missing fusion factors are supplied in the diluent. These results again suggest Uso1p function is independent from and precedes Sec18p and LMA1 action. Incubation with Uso1p alone followed by dilution with Sec18p and LMA1 (Fig. 10, ▪) was not the same as that observed for the complete reaction diluted with buffer containing ATP (Fig. 10, •). This was due to dilution of the Sec18p and LMA1 proteins under the latter condition whereas active concentrations of these fusion factors were maintained under the former. Therefore at early time points, Uso1p-docked vesicles efficiently chase due to maintenance of LMA1 and Sec18p throughout the second incubation but docked vesicles for the complete reaction (Fig. 10, •) were not chased as efficiently because of dilution of Sec18p and LMA1. At later times (20 and 30 min), the fusion efficiency of the Uso1p-docked intermediate was not as efficient as that observed with the complete reaction. This may be because of lability of the docked intermediate in the absence of Sec18p and LMA1.

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