Several ADP-ribosylation factor (Arf) isoforms support COPI vesicle formation

Abstract

Newly synthesized proteins and lipids are transported in vesicular carriers along the secretory pathway. Arfs (ADP-ribosylation factors), a family of highly conserved GTPases within the Ras superfamily, control recruitment of molecular coats to membranes, the initial step of coated vesicle biogenesis. Arf1 and coatomer constitute the minimal cytosolic machinery leading to COPI vesicle formation from Golgi membranes. Although some functional redundancies have been suggested, other Arf isoforms have been poorly analyzed in this context. In this study, we found that Arf1, Arf4, and Arf5, but not Arf3 and Arf6, associate with COPI vesicles generated in vitro from Golgi membranes and purified cytosol. Using recombinant myristoylated proteins, we show that Arf1, Arf4, and Arf5 each support COPI vesicle formation individually. Unexpectedly, we found that Arf3 could also mediate vesicle biogenesis. However, Arf3 was excluded from the vesicle fraction in the presence of the other isoforms, highlighting a functional competition between the different Arf members.

FIGURE 1.

Arf1, Arf3, Arf5, and Arf6 bind to Golgi membranes. Rat liver Golgi-enriched membranes were incubated with rat liver cytosol in the presence or absence of GTPγS and purified by centrifugation through two sucrose cushions. 6% of the initial reaction mix (I for Input) and 25% of primed-Golgi (pG) were subjected to SDS-PAGE and Western blot analysis.

FIGURE 2.

Arf1 and Arf5 but not Arf3 nor Arf6 are present in COPI vesicles. COPI vesicles were generated in vitro by adding rat liver cytosol to rat liver Golgi-enriched membranes in the presence or absence of GTPγS, and purified by centrifugation through two sucrose cushions. 1% of the initial reaction mix (I for Input) and 50% of the vesicle fraction (V) were subjected to SDS-PAGE and Western blot analysis.

FIGURE 3.

Arf4 and Arf5 are present in COPI vesicles. COPI vesicles were generated in vitro by adding rat liver cytosol to rat liver Golgi-enriched membranes in the presence or absence of GTPγS, and purified by centrifugation through two sucrose cushions. Samples were then submitted to peptide mass fingerprinting analysis. Arf4 and Arf5 were identified by multiple unique peptides (see “Experimental Procedures”): sequence coverage (bold red) obtained in both tryptic and chymotryptic digests (A and B) and fragmentation spectra of representative unique peptides for Arf4 (C) and Arf5 (D).

FIGURE 4.

Recombinant Arf1, Arf3, Arf4, Arf5, and Arf6 bind to Golgi-enriched membranes. Rat liver Golgi-enriched membranes were incubated with each of the recombinant Arf isoforms and purified rabbit liver coatomer in the presence or absence of GTPγS, and then purified by centrifugation through two sucrose cushions. 6% of the initial reaction mix (I for Input) and 25% of primed-Golgi (pG) were subjected to SDS-PAGE and Western blot analysis.

FIGURE 5.

Arf1, Arf3, Arf4, Arf5, but not Arf6 can generate COPI vesicle in vitro. COPI vesicles were generated in vitro from purified rabbit liver coatomer and recombinant myristoylated Arf1, Arf4, or Arf5 and rat liver Golgi-enriched membranes in the presence of GTPγS, and purified by centrifugation through two sucrose cushions. A, 1% of the initial reaction mix (I for Input) and 50% of the vesicle fraction (V) were subjected to SDS-PAGE and Western blot analysis. B, sample of the vesicle fractions were analyzed by negative stain electron microscopy. Scale bar: 250 nm.

FIGURE 6.

Quantification of Arf isoforms in rat liver cytosol. Defined aliquots of rat liver cytosol were subjected to SDS-gel electrophoresis and Western blotting. Blots were developed with antibodies directed against Arf isoforms as indicated, and quantified with reference to recombinant Arf isoforms. For details, see “Experimental Procedures.”

FIGURE 7.

Individual versus collective binding of Arf isoforms to Golgi membranes. Individual Arfs or a mixture of Arf1, Arf3, Arf4, and Arf5 were subjected according to their endogenous ratios (350 ng Arf1, 120 ng Arf3, 63 ng Arf4, 63 ng Arf5) to Golgi-enriched membranes and incubated for 10 min at 37 °C in the presence of GTPγS. Primed-Golgi was purified by centrifugation through two sucrose cushions. 6% of the initial reaction mix (I for Input) and 25% of primed-Golgi (pG) were subjected to SDS-PAGE and Western blot analysis.

FIGURE 8.

Vesicle preparation in the presence of individual or a mixture of Arf isoforms. Individual Arf or a mixture of Arf1, Arf3, Arf4 and Arf5 were subjected according to their endogenous ratios (2.1 μg Arf1, 0.72 μg Arf3, 0.38 μg Arf4, 0.38 μg Arf5) to Golgi-enriched membranes and incubated for 10 min at 37 °C in the presence of GTPγS. COPI vesicle were purified by centrifugation through two sucrose cushions. 1% of the initial reaction mix (I for Input) and 50% of the vesicle fraction (V) were subjected to SDS-PAGE and Western blot analysis.

FIGURE 9.

Characterization of the competition between Arf3 and the other Arf isoforms. A, Arf3 (0.72 μg) was mixed to each other Arf isoforms separately of all together (ArfMix) according to their endogenous ratios (2.1 μg Arf1, 0.38 μg Arf4, 0.38 μg Arf5) before being subjected to Golgi-enriched membranes and incubated for 10 min at 37 °C in the presence of GTPγS. COPI vesicles were purified by centrifugation through two sucrose cushions. 1% of the initial reaction mix (I for Input) and 50% of the vesicle fraction (V) were subjected to SDS-PAGE and Western blot analysis. In B–D, Arf3 (0.72 μg) has been mixed with Arf1 (B), Arf4 (C), and Arf5 (D) according to different ratios. Ratios are expressed as normalized values with regards to cytosol, i.e. a ratio of 1 corresponds to the endogenous ratio. For Arf4, normalized ratios have been calculated considering Arf4 and Arf5 to be expressed at similar levels.