COPII assay

Fig. 2. Image acquisition and processing steps to determine the transport of ts-045-G to the plasma membrane. HeLa cells were transfected with siRNAs on LabTek arrays as they are described in Chapter 1 of this issne. The ts-045-G transport assay was carried out as described in protocol 1.1 as described earlier in this chapter. Images were acquired sequentially using a lOX objective on a Scan R system using filters to detect specifically DAPI stained nuclei (A), Cy3 stained ts-045-G at the plasma membrane (B), and CFP-tagged ts-045-G (C). Images DT were generated as described in protocol 1.2. earlier in this chapter. R in (G) is the ratio of ts-045-G at the plasma membrane (measured in H) to ts-045-G expressed in cells (measured in I). Resnlts for siRNAs targeting the COPI component /3-COP, the COPII component Sec31p, and a p24 related membrane protein p26 are shown. The valnes are the average of two independent experiments (Bar = 50 /tm).

Fig. 2. Light scattering assay of the assembly of COPII coat with Secl2ACp and GTP. (A) The light scattering of a suspension of major-minor liposomes (100 ml ) in HKM buffer...

We thank Crystal Chan and Robert Lesch for COPII proteins and Matthew Welsh and David G. Drubin for sharing their equipment. We thank Chris Fromme for improving the manuscript and Bruno Antonny for advice on kinetic analysis of real-time assays. We thank... 

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.

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. 

The nature and the concentration of the monovalent salt used in the assay buffer are important parameters. We use Kacetate as we noticed less liposome recruitment of coatomer in experiments conducted with KCl or NaCl (see following). For the COPII coat, it is important to keep the ionic strength of the sample within 180-240 mM (taking into account the contribution of the COPII components). The Sec23/24 complex aggregates below 160 mM salt. 

The buffer used for the preparation of hposomes should be isoosmotic with the assay buffers in which proteins and hposomes are finally mixed. It is preferable to omit divalent salt (MgC ) in the liposome buffer to prevent long-term fusion. Therefore the liposomes used for experiments with COPI proteins are prepared in 50 mM Hepes-KOH, pH 7.2,120 mM Kacetate, whereas hposomes used for experiments with COPII proteins are prepared in 20 mM Hepes-KOH, pH 7.0, 160 mM Kacetate. 

PA is a minor component of the ER membrane that accounts for less than 1% of total ER membrane lipids (Allan, 1996). Formed PA is rapidly consumed by the activity of phosphatidate phosphohydrolase (PAP). In order to measure the formation of PA, the dynamics of PA formation and consumption has to be controlled. This is achieved by exploiting a unique transphosphatidylation reaction that is catalyzed by PLD enzymes. In this reaction, the aliphatic chain of a primary alcohol is transferred to the phosphatidyl moiety of the phosphatidic acid product. In the presence of low concentrations of primary alcohols, PLD enzymes generate phospha-tidylalcohols, which are not recognized by PAP and are not efficiently consumed (Morris et ah, 1997). Therefore the measurement of transphosphatidylation activity of PLD provides a convenient assay that avoids the otherwise highly dynamic nature of the lipid remodeling cascade induced by Sari to support COPII mediated ER export. 

Consistent with its ability to inhibit COPII budding in vivo, CI-976 has been found to inhibit the budding of COPII vesicles in an in vitro reconstitution assay. The procedures for conducting this assay are detailed elsewhere (Rowe et al, 1996), so we will describe our modifications for use with CI-976. 

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