Intramitochondrial distribution and transport of phosphatidylserine and its decarboxylation product, phosphatidylethanolamine. Application of pyrene-labeled species.
To investigate the mechanism of intramitochondrial translocation of phosphatidylserine and its decarboxylation product, phosphatidylethanolamine, the distribution of these lipids between the outer (OM) and inner (IM) mitochondrial membranes, as well as their transversal and lateral distribution in OM were studied. Fluorescent, pyrenyl derivatives of phosphatidylserine (PyrxPS) and phosphatidylethanolamine (PyrxPE) species were employed because they allow: (i), direct monitoring of PS (and PE) loading to the mitochondria; (ii) assay of PS decarboxylation by high-performance liquid chromatography with fluorescence detection and (iii), determination of the lateral distributions of PS and PE within the mitochondrial membranes. All PyrxPS species tested were efficiently decarboxylated by the solubilized decarboxylase and thus the distribution of the endogenous PE could be also studied. When the PyrxPS species were loaded to isolated mitochondria very little, if any, of the loaded PyrxPS or of the PyrxPE product was found in IM independent of the time and temperature of incubation, strongly suggesting that these lipids either never enter IM or their residence there is only transient. When mitochondria preloaded with Pyr4PS were incubated with an excess of acceptor vesicles in the presence of the lipid transfer protein, 80% of Pyr4PS and 30-40% of the Pyr4PE product were transported to the acceptor vesicles, indicating that at least corresponding fractions of these lipid were located in, or were in rapid equilibrium with the outer leaflet of OM. Since the decarboxylase is located in the inner membrane, these results signify that both PS and PE must be able to move readily across OM. Determination of the excimer to monomer ratio as the function of pyrenyl lipid concentration in mitochondria (i.e., OM) gave parallel results for PyrxPS and -PE species suggesting the lateral distribution of PS and PE in OM is similar and thus there is no specific enrichment of PS to the contact sites. To investigate the mechanism of PS transport from the outer leaflet to the decarboxylation site, the influence of PyrxPS hydrophobicity, i.e., pyrenylacyl chain length, on the rate of decarboxylation was determined. The variation of the length of the pyrenyl acyl chain from 4 to 12 carbons did not significantly affect the rate of PyrxPS decarboxylation in intact mitochondria, indicating that the transport of PS from the outer leaflet of OM to the site of decarboxylation takes place by lateral diffusion rather than by spontaneous or protein-mediated transport. The implications of these findings on the mechanism of intramitochondrial transport of PS and PE are discussed in terms of alternative models.
Department of Cellular Biochemistry, Nencki Institute on Experimental Biology, Warsaw, Poland.
R
R
Jasińska
J
J
Zborowski
P
P
Somerharju
D000818:Animals; D001692:Biological Transport; D002262:Carboxy-Lyases / Q000378:metabolism; D004058:Diffusion; D005453:Fluorescence; D007425:Intracellular Membranes / Q000737:chemistry / Q000378:metabolism*; D008928:Mitochondria / Q000378:metabolism*; D008954:Models, Biological; D010713:Phosphatidylcholines / Q000737:chemistry / Q000494:pharmacology; D010714:Phosphatidylethanolamines / Q000378:metabolism*; D010718:Phosphatidylserines / Q000378:metabolism*; D051381:Rats; D017208:Rats, Wistar
D010713:Phosphatidylcholines; D010714:Phosphatidylethanolamines; D010718:Phosphatidylserines; C021248:1-palmitoyl-2-pyrenedecanoylphosphatidylcholine; D002262:Carboxy-Lyases; C022353:phosphatidylserine decarboxylase