Studies on the Intracellular Localization of Acetyl-CoA Carboxylase

Abstract

Since its original discovery as a soluble enzyme (4) AAC The present work was performed to identify the sub-has been assumed to be located in the cytoplasm. Earcellular localization of hepatic acetyl-CoA carboxylase lier work concluded that the activity of the enzyme was (ACC). Cellular organelles involved in fatty acid oxida-not associated with subcellular particles (5), but later tion that contain a malonyl-CoA sensitive carnitine reports indicated that activity of ACC could be detected palmitoyltransferase (CPT) activity or that are linked in high speed precipitates of rat-liver homogenates (6-to the control of this activity were analysed for the 8) and in a so-called mitochondrial fraction of such hopresence of ACC. No significant amount of ACC was mogenates (9,10). Furthermore, permeabilization of is subject to modifi cation of that activity by several factors (12-15), and the avidin binding method is sensitive to interference by other biotin-containing proteins competing for labelled avidin (16). Therefore, the deterDepending on the physiological state of the animal, mination of subcellular distribution on the basis of avithe liver is a tissue that can either exhibit high rates din binding or enzyme activity is difficult to interpret. of fatty acid biosynthesis or high rates of fatty acidThe availability of ACC antibodies (17) and the techoxidation. Control of the activity of acetyl-CoA carbox-nique of permeabilizing isolated hepa tocytes with digylase (ACC) is of special interest in this respect because itonin (15) has permitted to re-address the issue of lothe product of ACC, malonyl-CoA, is not only a sub-calization of ACC and to cir cumvent some of the methstrate for the cytosolic process of long-chain fatty acid odological problems. synthesis but is also an inhibitor of the activity of carnitine palmitoyltransferase (CPT), an important pace-MATERIALS AND METHODS setting enzyme of long-chain fatty acid oxidation (1). As a matter of fact, malonyl-CoA sensitive CPT activity Male Wistar rats (250-300 g) which had free access to food and is present both in mitochondrial outer membranes water were used throughout in this study. Hepatocytes were isolated (CPT-I) (2) and in the peroxisomal matrix (3). and incubated as described in (18). Acetyl-CoA carboxylase activity, Given the functions of malonyl-CoA in or on different isoform distribution and mass were determined in isolated hepatic organelles of the cell, the enzyme responsible for its mitochondria, isolated hepatic peroxisomes or in digitonin-permeabilized hepatocytes. For isolation of mitochondria from liver tissue, production, ACC, may be compartmentalized as well.the procedure described by was followed. Mitochondria from hepatocytes were isolated by homogenizing cells (4-6 mg of protein) with a loose-fitting Dounce homogenizer in a low

Flock House Virus RNA Replicates on Outer Mitochondrial Membranes in Drosophila Cells

“…3A), similar to the distribution of VDAC, an outer mitochondrial integral membrane protein (52). In contrast, the cytoplasmic protein acetyl coenzyme A carboxylase (20) was recovered in the soluble fraction. We also investigated the membrane association of protein A under buffer conditions designed to remove peripheral proteins loosely associated with intracellular membranes (Fig.…”

“…Samples were separated by SDS-PAGE, transferred to a PVDF membrane, and immunoblotted. We analyzed partitioning of the 30-kDa outer mitochondrial membrane protein VDAC (52) and the 265-kDa cytoplasmic protein acetyl coenzyme A carboxylase (20) as controls for the pellet and soluble fractions, respectively. (B) Differential centrifugation of protein A from FHVinfected cell lysates after treatment with buffer conditions designed to dislodge peripherally associated membrane proteins or a nonionic detergent.…”

The subcellular localization of acetyl-CoA carboxylase 2

“…The concentration of malonyl-CoA and the activity of ACC2 in skeletal muscle, for instance, are controlled rapidly during contraction of the muscle fibers by diminishing ACC2 activity by increasing its phosphorylation through the action of kinases (10)(11)(12)(13)(14)(15)(16)(17)(18)36). A lower concentration of malonyl-CoA results in increasing CPT1 activity and, hence, in increased fatty acid oxidation and energy production, a condition needed in exercising animals (37).…”

Demonstrated and inferred metabolism associated with cytosolic lipid droplets

“…Early reports suggested that a fraction of mammalian acetyl-CoA carboxylase was associated with mitochondria or peroxisomes [references contained within ( 17 )] but later work did not confi rm these observations in rat liver; on the contrary, evidence involving cytoskeletal agents suggested that some of the enzyme was associated with microtubules ( 17 ). In S. cerevisiae , the protein was originally localized to the surface of the ER ( 18 ).…”