Paul François Gallet

Transbilayer movement and distribution of spin-labelled phospholipids in the inner mitochondrial membrane

The transmembrane diffusion and equilibrium distribution of spin-labelled phosphatidylethanolamine (PE*), phosphatidylcholine (PC*) and cardiolipin (CL*) were investigated in purified mitochondrial inner membranes using electron spin resonance spectroscopy. Using the back exchange technique, we found that the outside-inside movement of PE* and PC* in beef-heart inner mitochondrial membranes was rapid (t1/2 in the range 10-15 min at 30 degrees C). The steady-state distributions in non-energised mitoplasts were approximately 30% in the inner leaflet for PC* and 39% for PE*. Within the limits of probe concentration that can possibly be used in these experiments, the initial velocity of the inward movement was not saturable with respect to the amount of analogue added to the membranes, suggesting that the spin-labelled phospholipids diffused passively between the two leaflets of the inner mitochondrial membrane. In energised mitoplasts, PC* behaviour was not affected, PE* diffused approximately two times faster toward the inner monolayer but reached the same plateau. Treatment of energised mitochondria with N-ethylmaleimide did not affect PC* diffusion, while the kinetics of PE* internalisation became identical to that of PC*. Similar results were found when PC* and PE* movements were studied in mitoplasts from beef heart, rat liver or yeast. The spin-labelled cardiolipin, which possesses four long chains, had to be introduced in the mitoplast with some ethanol. After equilibration (t1/2 of the order of 13 min at 30 degrees C), the transmembrane distribution suggested that approximately half of the cardiolipin analogue remained in the outer leaflet. These results do not allow us to determine if a specific protein (or flippase) is involved in the phospholipid transmembrane traffic within inner mitochondrial membranes, but they show that lipids can rapidly flip through the mitochondrial membrane.

Chemo-enzymatic synthesis of a selectin ligand using recombinant yeast cells

Abstract Functional soluble human α(1-3/4) fucosyltransferase was successfully expressed in Pichia pastoris cells. The recombinant protein was located in the periplasmic space; thus, incubation of the whole yeast cells with disulfated tetrasaccharide1 (SO3−3)Galβ1-4(SO3−-6)GlcNAcβ1-3Galβ1-4GlcβOMBn and GDP-fucose directly provided pentasaccharide 2 (SO3−3)Galβ1-4(Fucα1-3)(SO3−-6)GlcNAcβ1-3Galβ1-4GlcβOMBn in 70% yield.

Glycosylation-related Gene Expression in Prion Diseases PrPSc ACCUMULATION IN SCRAPIE INFECTED GT1 CELLS DEPENDS ON β-1,4-LINKED GalNAc-4-SO4 HYPOSULFATION

Several lines of evidence indicate that some glycoconjugates are efficient effectors of the cellular prion protein (PrPC) conversion into its pathogenic (PrPSc) isoform. To assess how glycoconjugate glycan moieties participate in the biogenesis of PrPSc, an exhaustive comparative analysis of the expression of about 200 glycosylation-related genes was performed on prion-infected or not, hypothalamus-derived GT1 cells by hybridization of DNA microarrays, semiquantitative RT-PCR, and biochemical assays. A significant up- (30-fold) and down- (17-fold) regulation of the expression of the ChGn1 and Chst8 genes, respectively, was observed in prion-infected cells. ChGn1 and Chst8 are involved in the initiation of the synthesis of chondroitin sulfate and in the 4-O-sulfation of non-reducing N-acetylgalactosamine residues, respectively. A possible role for a hyposulfated chondroitin in PrPSc accumulation was evidenced at the protein level and by determination of chondroitin and heparan sulfate amounts. Treatment of Sc-GT1 cells with a heparan mimetic (HM2602) induced an important reduction of the amount of PrPSc, associated with a total reversion of the transcription pattern of the N-acetylgalactosamine-4-O-sulfotransferase 8. It suggests a link between the genetic control of 4-O-sulfation and PrPSc accumulation.