Identification and Characterization of Associated with Lipid Droplet Protein 1: A Novel Membrane‐Associated Protein That Resides on Hepatic Lipid Droplets

Abstract

Alcoholic and nonalcoholic liver steatosis and steatohepatitis are characterized by the massive accumulation of lipid droplets (LDs) in the cytosol of hepatocytes. Although LDs are ubiquitous and dynamic organelles found in the cells of a wide range of organisms, little is known about the mechanisms and sites of LD biogenesis. To examine the participation of these organelles in the pathophysiological disorders of steatotic livers, we used a combination of mass spectrometry (matrix-assisted laser desorption ionization-time of flight and LC-MS electrospray) and Western blot analysis to study the composition of LDs purified from rat liver after a partial hepatectomy. Fifty proteins were identified. Adipose differentiation-related protein was the most abundant, but other proteins such as calreticulin, TIP47, Sar1, Rab GTPases, Rho and actin were also found. In addition, we identified protein associated with lipid droplets I [ALDI (tentatively named Associated with LD protein 1), a novel protein widely expressed in liver and kidney corresponding to the product of 0610006F02Rik (GI:27229118). Our results show that, upon lipid loading of the cells, ALDI translocates from the endoplasmic reticulum into nascent LDs and indicate that ALDI may be targeted to the initial lipid deposits that eventually form these droplets. Moreover, we used ALDI expression studies to view other processes related to these droplets, such as LD biogenesis, and to analyze LD dynamics. In conclusion, here we report the composition of hepatic LDs and describe a novel bona fide LD-associated protein that may provide new insights into the mechanisms and sites of LD biogenesis.

A novel role for lipid droplets in the organismal antibacterial response

“…LDs were purified from murine liver using a previously established protocol (Turro et al, 2006); the hepatocyte lipid-droplet resident protein (Turro et al, 2006) ALDI was enriched 10 3 -fold (Figure 6A), confirming the success of the fractionation. By Western blotting, we detected histone H1 in the droplet fraction, using three different specific antibodies generated in different species (Figure 6B).…”

“…Male mice of approximately 8–12 weeks old were intraperitoneally injected with 20 ng LPS diluted in NaCl 0.9% or with an equivalent volume of NaCl and fasted for 16 hr. Hepatic LDs were isolated exactly as described in Turro et al, (2006) (Wan et al, 2007). The protein fraction of LDs was separated by precipitation with 33% of cold TCA and the resulting pellets resuspended in 50 μl of 250 mM Tris, 2% SDS.…”

“…The primary antibodies used in immunoblot analysis of mouse samples were anti-histone H1, clone AE-4 from UPSTATE biotechnology (Millipore, MA, USA), Anti histone H1: sc-10806, anti histone H3 (FL-136): sc-10809 and anti histone H2B (FL-126): sc-10808 (from Santa Cruz Biotechnology, CA, USA), anti histone H1.2, anti histone H2A and anti histone H2B (from Abcam, Cambridge, UK). The polyclonal anti-ALDI used in this study is described in Turro et al, 2006.…”

Annexins – insights from knockout mice

“…AnxA6 is predominantly found at the plasma membrane and endosomes (Grewal et al, 2005;Cubells et al, 2007;Vilà de Muga et al, 2009). Other AnxA6 locations include mitochondria, lipid droplets, and the secretory pathway (Gerke and Moss, 2002;Freye-Minks et al, 2003;Gerke et al, 2005;Turró et al, 2006;Chlystun et al, 2013). These various cellular locations, together with multiple interaction partners identified in tissues, cell culture and in vitro studies likely reflect the many different regulatory AnxA6 functions.…”

Not Just Fat: The Structure and Function of the Lipid Droplet

“…The bidirectional movement of some proteins between LDs and the ER membrane suggests a close structural relationship (Pol et al 2005;Turro et al 2006;Zehmer et al 2009). However, it has not been shown unequivocally that normal LDs retain membrane continuity with the ER as plastoglobules do with the thylakoid membrane ( Fig.…”