Stable expression of functional mitochondrial uncoupling protein in Chinese hamster ovary cells.

Abstract

The mitochondrial uncoupling protein (UCP) is a membranous proton carrier exclusively synthesized in brown adipocytes. The cDNA for the rat UCP was placed in an expression vector and transfected into mammalian cells. Its expression was tested in transiently transfected CHO cells. In these cells the UCP was detected in mitochondria by using antibodies. Permanent expression of the UCP was achieved in stable transformed CHO cell lines. In these cells the UCP was characterized in mitochondrial membranes, by using antibodies and hydroxyapatite purification. The protein expressed in CHO cells displayed the functional characteristics of brown adipocyte UCP. It induced the uncoupling of respiration in isolated CHO mitochondria. The membrane potential of transformed mitochondria was also significantiy lowered, as a result of the proton translocating activity of the UCP. GDP is known to inhibit the proton pathway in brown fat mitochondria. Addition of GDP to CHO mitochondria containing UCP resuited in a recoupling of respiration and an increase in membrane potential. Thus we conclude that functional UCP is expressed in CHO cells and that the insertion of the UCP alone in any mitochondria is sufficient to induce the uncoupling of respiration. This approach should allow studies on the structure-function relationship of the UCP and of several other related mitochondrial carriers.In most eukaryotic cells, the energetic equilibrium results from processes that take place in the inner mitochondrial membrane. In this cell compartment, the synthesis of ATP is coupled to the oxidation process, so that respiration ceases when no ADP and phosphate are available. This is why two mitochondrial transporters, the ADP/ATP carrier and the phosphate carrier, play a significant role in the control of oxidative phosphorylation. These two carriers are present in any cell. The uncoupling protein (UCP) is a mitochondrial transporter (1, 2) that is expressed only in brown adipocytes of mammals, where it induces energy dissipation as heat. UCP acts as a natural uncoupler ofoxidative phosphorylation capable of collapsing the proton electrochemical gradient necessary for ADP phosphorylation (1). Its activity is inhibited by purine nucleotides (1, 3, 4) and its synthesis is essentially regulated at the level of transcription by the sympathetic nervous system acting through norepinephrine released at the surface of brown adipocytes (2, 5). Interestingly, several laboratories have reported that UCP, the ADP/ATP carrier, the mitochondrial phosphate carrier, and a recently identified component named hML-7 belong to the same protein family (6-10) and were derived from a common ancestor. Moreover UCP, the ADP/ATP carrier, and hML-7 have no N-terminal targeting sequence.Studies on UCP offer the possibility of investigating several fundamental questions such as the mechanisms of proton transport by membrane carriers, the organization of a regulatory nucleotide binding site, the topology ofa mitochondrial carrier folded inside the membrane, and lastly...

Fatty acids do not activate UCP2 in pancreatic beta cells: comparison with UCP1

“…Previous studies have shown that the functional properties of UCP1 described in brown adipose tissue mitochondria in response to fatty acids [29] are also observed when the protein is expressed in other cell types like skeletal muscle cells [30] or CHO cells [31]. The present study shows that this is also the case when UCP1 is expressed in the pancreatic β-cell line INS-1.…”

Expression of the mitochondrial uncoupling protein gene from the aP2 gene promoter prevents genetic obesity.

“…The ability of UCP to function ectopically is not surprising, since it has been shown that mammalian UCP reconstituted into Chinese hamster ovary cells (24) or yeast mitochondria (25) is functional. The important point is that only very low levels of UCP in the white fat may be sufficient to affect obesity and that a broader spectrum of tissues and cell types may be capable of harboring a UCP based thermogenic system that could control obesity.…”

The uncoupling protein homologues: UCP1, UCP2, UCP3, StUCP and AtUCP

“…Mitochondria endowed with a variety of other carriers are more coupled than are BAT mitochondria. The introduction of UCP1 either in CHO cell mitochondria, or in yeast mitochondria using recombinant DNA technology, induced a partially uncoupled state [81,105]. When yeast mitochondria containing UCP1 were challenged with fatty acids, they showed a much higher sensitivity to the fatty acids than the control mitochondria [106].…”