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

Abstract

Proteins critical for vesicle formation by the Coat Protein I (COPI) complex are being identified, but less known has been the role of specific lipids. Brefeldin-A ADP-Ribosylated Substrate (BARS) acts in the fission step of COPI vesicle formation. Here, we show that BARS induces membrane curvature in cooperation with phosphatidic acid (PA). This revelation has allowed us to further delineate COPI vesicle fission into two sub-stages: i) an earlier stage of bud neck constriction, in which BARS and other COPI components are needed, and ii) a later stage of bud neck scission, in which PA generated by phospholipase D2 (PLD2) is needed additionally. Moreover, in contrast to the disruption of the Golgi seen upon perturbing the core COPI components (such as coatomer), inhibition of PLD2 results in milder disruptions, predicting that such COPI components have additional role(s) in maintaining Golgi structure other than through COPI vesicle formation.

CtBPs promote mitotic fidelity through their activities in the cell nucleus

“…CtBP chromatin-modifying complexes are readily detectable in proliferating cancer cells in culture (Shi et al, 2003), and there is evidence that their formation is increased under conditions of increased glycolysis, for example, in response to hypoxia (Zhang et al, 2002). The C-terminal domain makes secondary interactions with some PxDLS-containing factors (Kuppuswamy et al, 2008) and is also the minimal lipid interaction domain (Yang et al, 2008). Dimerization of CtBP molecules is required for the formation of an active chromatin-modifying complex (Kuppuswamy et al, 2008); we reasoned, therefore, that an N-terminal fragment of CtBP lacking the dimerization domain would act as a dominant negative by binding components of the CtBP repression complex and preventing their interaction with CtBP dimers.…”

“…Dimerization of CtBP molecules is required for the formation of an active chromatin-modifying complex (Kuppuswamy et al, 2008); we reasoned, therefore, that an N-terminal fragment of CtBP lacking the dimerization domain would act as a dominant negative by binding components of the CtBP repression complex and preventing their interaction with CtBP dimers. This region does not bind lipids (Yang et al, 2008), so would not be predicted to affect CtBP-dependent Golgi fission. The N terminus of CtBP2 was used, as this also contains a nuclear localization/retention motif (NLS, amino acids 8-13) .…”

“…We have also clearly demonstrated that CtBP function in the nucleus is critical for the regulation of mitotic fidelity, as are its interactions with cellular factors that associate with the PxDLS-binding cleft. Previous studies have successfully used a similar dominant-negative approach to probe the role of CtBPs in Golgi maintenance, for example, (Hidalgo Carcedo et al, 2004) in contrast to the approach we have used here, these studies included the C-terminal domain of CtBP in their dominant-negative constructs, which has the potential to disrupt the interaction of full length CtBPs with lipids, and hence inhibit CtBP-dependent Golgi fission (Yang et al, 2008).…”

Biochemical characterization of the interaction between HspA1A and phospholipids

“…It is known that binding of HspA1A to BMP and HspA8 to PS prevents permeabilization of the lysosomal membrane and mediates microautophagy by late endosomes, respectively Sahu et al 2011). Also, HspA1A binds to several lipids involved at various stages of endocytosis (PA, PS, BMP, PI(5)P, and PI(3,4,5)P 3 ) (McCallister et al 2015;van Meer et al 2008;Yang et al 2008). Therefore, the ability of HspA1A to associate with lipids may allow the protein to participate in multiple stages of the endocytic pathway, e.g., vesicle formation, targeting, and fusion, which require precise regulation and numerous changes in lipid composition (Cho and Stahelin 2005;Yang et al 2008).…”

“…Also, HspA1A binds to several lipids involved at various stages of endocytosis (PA, PS, BMP, PI(5)P, and PI(3,4,5)P 3 ) (McCallister et al 2015;van Meer et al 2008;Yang et al 2008). Therefore, the ability of HspA1A to associate with lipids may allow the protein to participate in multiple stages of the endocytic pathway, e.g., vesicle formation, targeting, and fusion, which require precise regulation and numerous changes in lipid composition (Cho and Stahelin 2005;Yang et al 2008). HspA1A may also interact with proteins that will be released, e.g., PrPC, a GPI anchor protein, and enhance their membrane localization and release (Wang et al 2011).…”