A role for phosphatidic acid in COPI vesicle fission yields insights into Golgi maintenance

Abstract

Proteins essential for vesicle formation by the Coat Protein I (COPI) complex are being identified, but less is known about the role of specific lipids. Brefeldin-A ADP-ribosylated substrate (BARS) functions in the fission step of COPI vesicle formation. Here, we show that BARS induces membrane curvature in cooperation with phosphatidic acid. This finding has allowed us to further delineate COPI vesicle fission into two sub-stages: 1) an earlier stage of bud-neck constriction, in which BARS and other COPI components are required, and 2) a later stage of bud-neck scission, in which phosphatidic acid generated by phospholipase D2 (PLD2) is also required. Moreover, in contrast to the disruption of the Golgi seen on perturbing the core COPI components (such as coatomer), inhibition of PLD2 causes milder disruptions, suggesting that such COPI components have additional roles in maintaining Golgi structure other than through COPI vesicle formation.

Figure 1. Liposome tubulation by BARS requires PA

(A) Binding by BARS to liposomes with increasing level of additional specified lipid. (B) EM examination of liposomes bound by BARS; bar, 200 nm. The fraction that exhibited tubulation was quantified, with mean and standard error from three experiments shown. (C) Quantitation for the ability of other proteins to induce liposome tubulation, as analyzed by EM.

Figure 2. Perturbation of PA generated by PLD2 affects COPI transport

(A) Gel shows knockdown of proteins (upper panels) with loading controls (lower panels). Graph shows quantitation of retrograde transport of the chimeric KDELR, with the mean from three experiments and standard error shown. (B) Gel shows knockdown of proteins. Graph again shows quantitation of retrograde transport of the chimeric KDELR. (C) Quantitation of retrograde transport of the chimeric KDELR upon microinjection of different antibodies in conditions, with the mean from three experiments and standard error shown. (D) First stage shows ARF1-dependent recruitment of coatomer to Golgi membrane (above). Second stage shows COPI vesicle formation, as reflected by the release of coatomer from Golgi membrane (below).

Figure 3. COPI vesicle formation requires PA generated by PLD2

(A) Vesicle formation as reflected by the release of β–COP from Golgi membrane at the second stage. (B) Quantitation of vesicle formation as reflected by the release of β–COP from Golgi membrane at the second stage. The mean from three experiments with standard error is shown. (C) Vesicle formation as reflected by the release of β–COP from Golgi membrane at the second stage. (D) Cargo sorting into COPI vesicles, as reflected by the release of cargo proteins from Golgi membrane at the second stage. (E) Vesicle formation assessed by immunogold EM. The mean from three experiments with standard error is shown.

Figure 4. PLD2 is required for at a late stage of COPI vesicle fission

(A) EM examination of Golgi membrane after the second stage; bar, 50 nm. As comparison, Golgi membrane prior to the incubation is shown as control. (B) The distributions of proteins on Golgi membrane after the second stage as assessed by immunogold EM; bar, 50 nm. Quantitation with the mean from three experiments and standard error is also shown. (C) Three-dimensional reconstruction of a representative Golgi from either control or PLD2-depleted cells is shown in two opposite views. Yellow highlights buds. (D) Quantitation of buds accumulation. The mean and standard deviation, derived from analyzing ten randomly selected Golgi profiles in each condition, are shown.

Figure 5. Golgi morphology and COPI distribution upon PLD2 depletion

(A) Relative distributions of COPI labeling at the Golgi. The mean from ten randomly selected Golgi profiles with standard deviation is shown. (B) Golgi in control cells. (C-E) Golgi in PLD2-depleted cells; bar, 200 nm.