INTRAMITOCHONDRIAL DISTRIBUTION AND TRANSPORT OF PHOSPHATIDYLSERINE AND ITS DECARBOXYLATION PRODUCT, PHOSPHATIDYLETHANOLAMINE - APPLICATIONOF PYRENE-LABELED SPECIES

To investigate the mechanism of intramitochondrial translocation of ph osphatidylserine and its decarboxylation product, phosphatidylethanola mine, the distribution of these lipids between the outer (OM) and inne r (IM) mitochondrial membranes, as well as their transversal and later al distribution in OM were studied. Fluorescent, pyrenyl derivatives o f phosphatidylserine (Pyr(x)PS) and phosphatidylethanolamine (Pyr(x)PE ) species were employed because they allow: (i), direct monitoring of PS (and PE) loading to the mitochondria; (ii), assay of PS decarboxyla tion by high-performance liquid chromatography with fluorescence detec tion and (iii), determination of the lateral distributions of PS and P E within the mitochondrial membranes. All Pyr(x)PS species tested were efficiently decarboxylated by the solubilized decarboxylase and thus the distribution of the endogenous PE could be also studied. When the Pyr(x)PS species were loaded to isolated mitochondria very little, if any, of the loaded Pyr(x)PS or of the Pyr(x)PE product was found in IM independent of the time and temperature of incubation, strongly sugge sting that these lipids either never enter IM or their residence there is only transient. When mitochondria preloaded with Pyr4PS were incub ated 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 corresp onding fractions of these lipid were located in, or were in rapid equi librium with the outer leaflet of OM. Since the decarboxylase is locat ed in the inner membrane, these results signify that both PS and PE mu st be able to move readily across OM. Determination of the excimer to monomer ratio as the function of pyrenyl lipid concentration in mitoch ondria (i.e., OM) gave parallel results for Pyr(x)PS and -PE species s uggesting the lateral distribution of PS and PE in OM is similar and t hus there is no specific enrichment of PS to the contact sites. To inv estigate the mechanism of PS transport from the outer leaflet to the d ecarboxylation site, the influence of Pyr(x)PS hydrophobicity, i.e., p yrenylacyl chain length, on the rate of decarboxylation was determined . The variation of the length of the pyrenyl acyl chain from 4 to 12 c arbons did not significantly affect the rate of Pyr(x)PS decarboxylati on in intact mitochondria, indicating that the transport of PS from th e outer leaflet of OM to the site of decarboxylation takes place by la teral diffusion rather than by spontaneous or protein-mediated transpo rt. The implications of these findings on the mechanism of intramitoch ondrial transport of PS and PE are discussed in terms of alternative m odels.