Figure 1.

Cytoplasmic mobility of COPI proteins. Representative fluorescence-correlation spectroscopy (FCS) curves for (A) green fluorescent protein (GFP) in buffer and cytoplasm, (B) ADP ribosylation factor 1 (ARF1), (C) ARF GTPase-activating protein 1 (ARFGAP1) and (D) ε-COP (ε-coat-protein) in HeLa cells (black lines), with best fits according to equation (1) (red symbols; see Methods). The autocorrelation function C(τ) shown for ARF1 involves a fast (87%; D ≈ 15 μm² s⁻¹; where D is the diffusion coefficient) and a slow (13%; D ≈ 0.5 μm² s⁻¹) population, which is probably because of interactions with nucleotide-exchange factors. Similarly, ARFGAP1 shows a large, fast pool (40%; D ≈ 13 μm² s⁻¹) and a minority of slow molecules (60%; D ≈ 0.5 μm² s⁻¹), which increased on addition of brefeldin A (BFA). The FCS curve of ε-COP comprises a fast component (73%; D ≈ 16 μm² s⁻¹), due to monomeric ε -COP, and a slow one (27%; D ≈ 0.5 μm² s⁻¹), due to ε -COP, which is incorporated into coatomer. (E) In untreated Chinese hamster ovary (CHO) cells, the FCS curve also showed two components (left curve, fast, 45%; D ≈ 16 μm² s⁻¹; slow, 55%; D ≈ 0.5 μm² s⁻¹), and applying BFA to release coatomer from Golgi membranes increased the slow component (to 68%). (F) Knocking out the β-COP subunit of coatomer reduced the slow component to less than 20%. This confirms that the slow component is the coatomer complex. RNAi, RNA intereference.