The liver isoform of carnitine palmitoyltransferase 1 is not targeted to the endoplasmic reticulum

Abstract

Liver microsomal fractions contain a malonyl-CoA-inhibitable carnitine acyltransferase (CAT) activity. It has been proposed [Fraser, Corstorphine, Price and Zammit (1999) FEBS Lett. 446, 69-74] that this microsomal CAT activity is due to the liver form of carnitine palmitoyltransferase 1 (L-CPT1) being targeted to the endoplasmic reticulum (ER) membrane as well as to mitochondria, possibly by an N-terminal signal sequence [Cohen, Guillerault, Girard and Prip-Buus (2001) J. Biol. Chem. 276, 5403-5411]. COS-1 cells were transiently transfected to express a fusion protein in which enhanced green fluorescent protein was fused to the C-terminus of L-CPT1. Confocal microscopy showed that this fusion protein was localized to mitochondria, and possibly to peroxisomes, but not to the ER. cDNAs corresponding to truncated (amino acids 1-328) or full-length L-CPT1 were transcribed and translated in the presence of canine pancreatic microsomes. However, there was no evidence of authentic insertion of CPT1 into the ER membrane. Rat liver microsomal fractions purified by sucrose-density-gradient centrifugation contained an 88 kDa protein (p88) which was recognized by an anti-L-CPT1 antibody and by 2,4-dinitrophenol-etomoxiryl-CoA, a covalent inhibitor of L-CPT1. Abundance of p88 and malonyl-CoA-inhibitable CAT activity were increased approx. 3-fold by starvation for 24 h. Deoxycholate solubilized p88 and malonyl-CoA-inhibitable CAT activity from microsomes to approximately the same extent. The microsomal fraction contained porin, which, relative to total protein, was as abundant as in crude mitochondrial outer membranes fractions. It is concluded that L-CPT1 is not targeted to the ER membrane and that malonyl-CoA CAT in microsomal fractions is L-CPT1 that is derived from mitochondria, possibly from membrane contact sites.

The Human Hydroxyacylglutathione Hydrolase (HAGH) Gene Encodes Both Cytosolic and Mitochondrial Forms of Glyoxalase II

“…To determine whether the subcompartments of the mitochondria can be adequately resolved by confocal fluorescence microscopy, pGII-DsRed2 was co-expressed with GFP-tagged carnitine palmitoyltransferase I (CPT-EGFP), a fusion protein previously used as an outer membrane marker (20). In other experiments, cells expressing pGIIDsRed2 were immunostained for cytochrome c, a marker previously used to visualize the intermembrane space (21).…”

“…Cells were transfected with pGII-DsRed2 alone or were co-transfected with a carnitine palmitoyltransferase I-EGFP fusion construct (20) or with plasmid pEYFP-Mito (Clontech) (400 ng total DNA/well in each case). After 72 h cells were fixed for 30 min with 4% paraformaldeyde in Dulbecco's modified phosphate-buffered saline (Sigma), washed twice with saline (10 min each), and mounted using Vectashield Hard Set (Vector Laboratories).…”

“…B, HepG2 cells were co-transfected with the pGII-DsRed2 fusion construct and an outer mitochondrial membrane marker (CPT1-EGFP described in Ref. 20). At default pinhole settings, partial colocalization of the signals was apparent (data not shown).…”

Membrane transport of fatty acylcarnitine and free l‐carnitine by rat liver microsomes

“…It has been envisaged that the substrate for this luminal CAT is fatty acylcarnitine, which is transported from the cytosol to the ER lumen [2,3,12] (Fig. 1) and which is generated by CPT 1 located at sites distinct from the ER [20] or also by an ER-targeted form of CPT 1 [12,[21][22][23]. For the luminal CAT to function in the way envisaged in Fig.…”

“…It has been envisaged that the substrate for this luminal CAT is fatty acylcarnitine, which is transported from the cytosol to the ER lumen [2,3,12] (Fig. 1) and which is generated by CPT 1 located at sites distinct from the ER [20] …”