Differential Regulation of Endoplasmic Reticulum Structure through VAP-Nir Protein Interaction

Abstract

The endoplasmic reticulum (ER) exhibits a characteristic tubular structure that is dynamically rearranged in response to specific physiological demands. However, the mechanisms by which the ER maintains its characteristic structure are largely unknown. Here we show that the integral ER-membrane protein VAP-B causes a striking rearrangement of the ER through interaction with the Nir2 and Nir3 proteins. We provide evidence that Nir (Nir1, Nir2, and Nir3)-VAP-B interactions are mediated through the conserved FFAT (two phenylalanines (FF) in acidic tract) motif present in Nir proteins. However, each interaction affects the structural integrity of the ER differently. Whereas the Nir2-VAP-B interaction induces the formation of stacked ER membrane arrays, the Nir3-VAP-B interaction leads to a gross remodeling of the ER and the bundling of thick microtubules along the altered ER membranes. In contrast, the Nir1-VAP-B interaction has no apparent effect on ER structure. We also show that the Nir2-VAP-B interaction attenuates protein export from the ER. These results demonstrate new mechanisms for the regulation of ER structure, all of which are mediated through interaction with an identical integral ER-membrane protein. The endoplasmic reticulum (ER)1 is an extensive network of membranes comprised of an array of interconnecting tubules and cisternae that emerges from the nuclear envelope (NE) and extends peripherally throughout the cell cytoplasm (1). It contains several structurally distinct domains, including the NE, the rough and smooth ER (rER and sER), and the regions that contact other organelles, such as the Golgi apparatus, the late endosomes, the lysosomes, mitochondria, peroxisomes, and the plasma membrane (2). The ER functions in diverse metabolic processes including lipid synthesis, carbohydrate metabolism, and the detoxification of drugs. It is responsible for the synthesis, translocation, glycosylation, folding, assembly, and processing of secretory and membrane proteins, and it functions in intracellular calcium storage and sequestering (3, 4).While the function of the ER in membrane trafficking, lipid biosynthesis, and calcium signaling have been extensively studied, the mechanism by which the ER maintains its characteristic structure in vivo remains largely unknown. Studies from yeast and mammalian cells have shown that the size and/or structure of the ER is extremely sensitive to certain cellular stress conditions, such as the unfolded protein response (UPR) or to the overexpression of a subset of ERresident membrane proteins (5, 6). Overexpression of 3-hydroxymethyl-3-methylglutaryl coenzyme A (HMG-CoA) reductase, microsomal aldehyde dehydrogenase (msALDH), cytochrome P-450, and malfolded cytochrome P-450, causes the proliferation of ER membranes and stacking of the ER cisternae into organized structures known as crystalloid ER, sinusoidal ER, or karmellae (7-12). The formation of these structures is easily visible and can be used as a quantitative method for studying ER membrane biogenesis (5). Neverth...

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

“…However, exposure of Nir2-depleted cells to 25OH resulted in cytosolic distribution of GFP-PH-OSBP, suggesting that under these conditions, Nir2 is crucial for PI4P production in the Golgi. Accordingly, we overexpressed either the wild-type Nir2 or Nir2 mutants truncated of the PI-transfer domain (⌬PI) or mutated in the FFAT motif (Amarilio et al, 2005) in VAP-depleted cells, and analyzed the subcellular localization of ␥-adaptin, OSBP, and CERT in cells grown in the presence of 25OH. The results clearly demonstrate the ability of wild-type Nir2, as well as the FFAT mutant, to restore the targeting of ␥-adaptin ( Figure 6A), OSBP ( Figure 6B), and CERT ( Figure 6C) to the Golgi of VAP-depleted cells.…”

“…The cells were lysed in lysis buffer (10 mM sodium phosphate, pH 7.4, 150 mM NaCl, 5 mM KCl, 2 mM EDTA, 2 mM EGTA, 0.5% Triton X-100, and protease inhibitors) and centrifuged at 16,000 ϫ g for 15 min at 4°C. VAP proteins were immunoprecipitated from the supernatants using the anti-VAPs antibody (Amarilio et al, 2005). The immunoprecipitates were washed three times with lysis buffer and boiled for 5 min in sample buffer containing 50 mM dithiothreitol, to cleave the cross-linker.…”

“…DNA constructs encoding either the wild-type Nir2-HA and its mutants, and the PI-transfer domain (amino acids [aa] 1-277), or the VAP-B-Myc, as well as antibodies against Nir2 and VAP were described previously (Amarilio et al, 2005;Litvak et al, 2005). Polyclonal antibodies against GRASP65 were raised in rabbits against glutathione-S-transferase fusion protein consisting amino acids 240-440 of the human GRASP65.…”

“…Previous studies have shown that VAPs interact with the FFAT-motif-containing proteins Nir2 (Amarilio et al, 2005), oxysterol-binding protein (OSBP) (Wyles et al, 2002), and CERT (Kawano et al, 2006). These three LT/BPs, which shuttle between the cytosol and the Golgi complex, are potentially functionally interdependent: CERT transports ceramide from the ER to the Golgi, and ceramide and PC are converted into sphingomyelin (SM) and diacylglycerol (DAG) by SM synthase (SMS) in the trans-Golgi (Hanada et al, 2007).…”

“…VAPs recruit FFAT-motif-containing proteins to the cytosolic surface of the ER membranes, through a conserved region within their MSP domain (Kaiser et al, 2005), and they have been implicated in regulation of membrane transport, phospholipid biosynthesis, and the unfolded protein response. Although their role in maintaining the identities of intracellular organelles has not been demonstrated, their ability to interact with lipid-transfer/binding proteins (LT/BPs) (Loewen et al, 2003) may affect the lipid composition of certain cellular membranes.Previous studies have shown that VAPs interact with the FFAT-motif-containing proteins Nir2 (Amarilio et al, 2005), oxysterol-binding protein (OSBP) (Wyles et al, 2002), and CERT (Kawano et al, 2006). These three LT/BPs, which shuttle between the cytosol and the Golgi complex, are potentially functionally interdependent: CERT transports ceramide from the ER to the Golgi, and ceramide and PC are converted into sphingomyelin (SM) and diacylglycerol (DAG) by SM synthase (SMS) in the trans-Golgi (Hanada et al, 2007).…”