Coordinated Lipid Transfer between the Endoplasmic Reticulum and the Golgi Complex Requires the VAP Proteins and Is Essential for Golgi-mediated Transport

Abstract

Lipid transport between intracellular organelles is mediated by vesicular and nonvesicular transport mechanisms and is critical for maintaining the identities of different cellular membranes. Nonvesicular lipid transport between the endoplasmic reticulum (ER) and the Golgi complex has been proposed to affect the lipid composition of the Golgi membranes. Here, we show that the integral ER-membrane proteins VAP-A and VAP-B affect the structural and functional integrity of the Golgi complex. Depletion of VAPs by RNA interference reduces the levels of phosphatidylinositol-4-phosphate (PI4P), diacylglycerol, and sphingomyelin in the Golgi membranes, and it leads to substantial inhibition of Golgimediated transport events. These effects are coordinately mediated by the lipid-transfer/binding proteins Nir2, oxysterolbinding protein (OSBP), and ceramide-transfer protein (CERT), which interact with VAPs via their FFAT motif. The effect of VAPs on PI4P levels is mediated by the phosphatidylinositol/phosphatidylcholine transfer protein Nir2, which is required for Golgi targeting of OSBP and CERT and the subsequent production of diacylglycerol and sphingomyelin. We propose that Nir2, OSBP, and CERT function coordinately at the ER-Golgi membrane contact sites, thereby affecting the lipid composition of the Golgi membranes and consequently their structural and functional identities. INTRODUCTIONThe unique lipid compositions of the secretory and endocytic organelles are critical for maintaining their distinct structural and functional identities (van Meer, 2000). Their identities are maintained despite extensive interconnecting vesicular-trafficking pathways, and they require tight regulation of lipid sorting, metabolism, and transport (van Meer, 1993;Pomorski et al., 2001;van Meer and Sprong, 2004). Increasing lines of evidence suggest that lipid-transfer proteins (LTPs) play a major role in regulating the lipid composition of membranous organelles (Sprong et al., 2001;De Matteis et al., 2007). These proteins facilitate lipid transfer between a donor and acceptor membrane (Holthuis and Levine, 2005), and they usually contain dual-targeting determinants for different membrane compartments. LTPs have been proposed to efficiently transfer lipids at membrane contact sites (MCSs) (Holthuis and Levine, 2005;Levine and Loewen, 2006).MCSs or zones of close apposition between the ER membranes and other cellular membranes, including the plasma membrane (PM), the membranes of the vacuoles, mitochondria, peroxisomes, lipid droplets, late endosomes, lysosomes, and the Golgi apparatus, have been identified in all eukaryotes by morphological and biochemical studies (Shore and Tata, 1977;Levine, 2004;Levine and Loewen, 2006). More recently, electron tomography studies have identified close contacts (10 -20 nm) between the ER and the outer mitochondrial membrane (Perkins et al., 1997) and between a specialized trans-ER and the three trans-most cisternae of the Golgi complex (Ladinsky et al., 1999;Marsh et al., 2001Marsh et al., , 2004. This ...

Role of Phosphatidylinositol 4-Phosphate (PI4P) and Its Binding Protein GOLPH3 in Hepatitis C Virus Secretion

“…Nonvesicular lipid transport between the ER and Golgi affects the lipid composition of the Golgi membranes (46). We and others (34,52) have shown that Golgi-associated OSBP and CERT are required for HCV virion secretion.…”

“…The PI4P-binding proteins OSBP and CERT that function in the Golgi trafficking were shown to affect HCV secretion (34,35,46,47). To investigate the functional role of Golgi-localized PI4P in the HCV secretion process, we used a mutant version of the hSac1 protein, hSac1 K2A.…”

Oxysterol Binding Protein–related Protein 9 (ORP9) Is a Cholesterol Transfer Protein That Regulates Golgi Structure and Function

“…Analysis of the binding specificity of the ORP9 and OSBP PH domains, both in isolation and in the context of the full-length protein, revealed a preference for monophosphorylated PIs, most notably PI-4P. Interaction of the OSBP PH domain with the Golgi apparatus is dependent on PI-4P synthesized by the PI 4-kinases III␤ (Balla et al, 2005) and/or II␣ (Wang et al, 2003), as well as other factors such as ARF (Levine and Munro, 2002) and Nir2 (Peretti et al, 2008). ORP9L did not localize with PI-4 kinase III␤ at the Golgi apparatus, and RNAi experiments showed that this kinase was not required for ORP9L localization (results not shown).…”

Endoplasmic Reticulum Structure and Interconnections with Other Organelles

“…4D) (Ladinsky et al 1999;Mogelsvang et al 2004). Depletion of integral ER membrane proteins VAP-A and VAP-B, resulted in mislocalization of Golgi lipid sensing and transport proteins (Nir2, OSBP, and CERT), and altered Golgi structure; furthermore, Nir2 was shown to directly transfer PI from the ER to the Golgi (Peretti et al 2008). Additional work shows the role of ceramide transport protein (CERT) in the nonvesicular transport of ceramide (a sphingomyelin precursor) from the ER to the trans-Golgi (Hanada 2010).…”