COPII components

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).

If the results of FRET experiments indicate that COPII components and SNARE can associate, it is important to show that the FRET signal is the result of a specific interaction. This can be demonstrated by showing that FRET does not occur between YFP-Sec24/23p and CFP-tagged unrelated membrane protein. We have performed FRET experiments using the ER resident protein Ufelp. Proteohposomes reconstituted with CFP-tagged form of MBP-Ufelp (Sato and Nakano, 2005) fails to produce an increase in FRET in the presence of YFP-Sec24/23p and Sarlp-GTP (or GMP-PNP) (Fig. 3B). 

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. 

Belden, W. J., and Barlowe, C. (1996). Erv25p, a component of COPII-coated vesicles, forms a complex with Emp24p that is required for efficient endoplasmic reticulum to Golgi transport. / Biol. Chem. 271, 26939-26946. 

A EXPERIMENTAL FIGURE 17-8 Vesicle buds can be visualized during in vitro budding reactions. When purified COPII coat components are incubated with isolated ER vesicles or artificial phospholipid vesicles (liposomes), polymerization of the coat proteins on the vesicle surface induces emergence of highly curved buds. In this electron micrograph of an in vitro budding reaction, note the distinct membrane coat, visible as a dark protein layer, present on the vesicle buds. [From K. Matsuoka etal., 1988, Ce//93(2) 263.[... 

COPII coats comprise three components the small GTP-blndlng protein Sari, a Sec23/Sec24 complex, and a Secl3/Sec31 complex. 

Salama, N. R., Chuang, J. S., and Schekman, R. W. (1997). Sec31 encodes an essential component of the COPII coat required for transport vesicle budding from the endoplasmic reticulum. Mol Biol Cell 8, 205-217. 

COPI and COPII vesicles mediate anterograde or retrograde traffic between the endoplasmic reticulum and the Golgi apparatus (Lee et al, 2004). Generation of COP vesicles is a multi-event process that starts with the recruitment of small G-proteins and large coat complexes on the Golgi or the endoplasmic reticulum (ER) membrane. At the membrane surface, coat proteins collect transmembrane proteins and polymerize into a curved lattice. The lattice shapes the underlying membrane into a bud, which by membrane fission leads to the formation of an individual transport vesicle. After vesicle formation, the coat depolymerizes and the COP components are recycled in the cytosol for another round. 

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. 

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. 

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