COPII assembly-disassembly assays

Fig. 3. Assembly/disassembly kinetics of COPII coat complex as detected by FRET and tryptophan fluorescence assay of GAP-catalyzed GTP hydrolysis on Sarlp with proteoliposomes. The reaction initially contained proteoliposomes (40 /rg Upids/ml) reconstituted with CFP-Betlp (60 nM) (A) or CFP-MBP-Ufelp (65 nM) (B) and Sarlp (830 nAf) loaded with 0.1 mM of GDP, GTP or GMP-PNP. After 10-min incnbation, YFP-Sec24/23p (160 nM) was added and the FRET signal at 530 nm was continnously monitored at 25°. (C) Tryptophan fluorescence assay of Sarlp GTP hydrolysis with CFP-Betlp reconstituted proteoliposomes. Tryptophan fluorescence was measured by the same procedure as (A), except that fluorescence was recorded at 340 nm upon excitation at 298 nm.

The assembly-disassembly cycle of CO PI and COPII coats is controlled by the GTPase cycle of the small G proteins Arfl and Sar. We describe here two spectroscopic assays that enable real-time studies of some elementary steps of coat assembly and disassembly on artificial liposomes of defined composition and curvature. A flotation assay to assess the effect of membrane curvature on protein adsorption to liposomes is also presented. 

Despite the overall complexity of coat assembly and vesicle formation, major advances have been made in the understanding of COP machineries. One breakthrough was the reconstitution of COPI and COPII assembly using purified components and artificial liposomes of defined composition (Bremser et al, 1999 Matsuoka et ah, 1998 Spang et al, 1998). In this chapter we describe two spectroscopic assays that complement the biochemical reconstitution and that enable the study of some dynamics aspects of protein coats, notably their assembly-disassembly cycle under the control of the small G-proteins Arf and Sar. In addition a biochemical flotation assay is detailed that permits fair determination of protein binding to liposomes of increasing curvature. 

Fig. 1. Schematic of FRET assay for monitoring the kinetics of COPII coat complex assembly and disassembly on SNARE reconstituted proteoliposomes. Sarlp-GTP leads to association of YFP-Sec24/23p with CFP-SNARE on proteoliposomes, with an accompanying increase in FRET between CFP and YFP. Subsequent GTP hydrolysis on Sarlp reverses the assembly process.

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