Some cultured cells contain significant amounts of a rarely recognized phospholipid, phosphatidylthreonine. Since phosphatidylthreonine is a structural analog of phosphatidylserine, the question rises whether it is transported to mitochondria and decarboxylated to phosphatidylisopropanolamine therein. We studied this issue with hamster kidney cell-line using a novel approach, i.e. electrospray mass-spectrometry and stable isotope-labeled precursors. Scanning for a neutral loss of 155, which is characteristic for phosphatidylisopropanolamine, indicated that this lipid is indeed present. The identity of phosphatidylisopropanolamine was supported by the following: (i) it cochromatographed with phosphatidylethanolamine; (ii) its molecular species profile was similar to that of phosphatidylethanolamine; (iii) its head group was labeled from 13 C-threonine; and (iv) its concentration increased in parallel with phosphatidylthreonine. Tests with solubilized decarboxylase and subcellular fractionation studies indicated that the low cellular content of phosphatidylisopropanolamine is due to inefficient decarboxylation, rather than poor translocation of phosphatidylthreonine to mitochondria. Importantly, the average hydrophobicity of phosphatidylisopropanolamine molecular species was significantly less than that of phosphatidylthreonine species, indicating that hydrophilic phosphatidylthreonine species translocate to mitochondria far more rapidly than hydrophobic ones. Parallel results were obtained for phosphatidylserine. These findings imply that efflux from the ER membrane could be the rate-limiting step in the phosphatidylthreonine and -serine translocation to mitochondria.Key words: BHK cells, electrospray, HPLC, lipid trafficking, MAM, mass-spectrometry, mechanism, mitochondria, neutral loss, phosphatidylserine, spontaneous transfer Received 21 December 2001, revised and accepted for publication 22 February 2002In cultured mammalian cells, most of phosphatidylethanolamine (PE) derives from phosphatidylserine (PS) via decarboxylation in mitochondria [reviewed in (1,2)]. Since PS is made in the ER, it has to translocate to mitochondria before 367 the decarboxylation can take place. This translocation is ATPdependent and seems to require close proximity of the ER (or rather a special region of ER membrane termed mitochondria-associated membrane, MAM) with the mitochondrial outer membrane (3), but the translocation mechanism has not been established. Recently, evidence was obtained indicating that the rate of translocation of newly synthesized PS to mitochondria is inversely related to molecular hydrophobicity, i.e. the less hydrophobic species (those with shorter and/or more unsaturated acyl chains) translocate to mitochondria more rapidly than the more hydrophobic species (4). This finding suggests that efflux of PS molecules from the ER membrane is the rate-limiting step in the translocation process. The simplest explanation for this would be that PS moves to mitochondria by spontaneous monomer diffusion via the cytop...
In baby hamster kidney and other cultured cells the majority of phosphatidylethanolamine (PE) is synthesized from phosphatidylserine (PS) in a process which involves transport of PS from the endoplasmic reticulum to mitochondria and decarboxylation therein by PS decarboxylase. To study the mechanism of this transport process, we first determined the molecular species composition of PE and PS from baby hamster kidney and Chinese hamster ovary cells. Interestingly, the hydrophilic diacyl molecular species were found to be much more abundant in PE than in PS, suggesting that hydrophilic PS species may be more readily transported to mitochondria than the hydrophobic ones. To study this, we compared the rates of decarboxylation of different PS molecular species in these cells. The cells were pulse labeled with [ 3 H]serine whereafter the distribution of the labels among PS and PE molecular species was determined by reverse phase high performance liquid chromatography and liquid scintillation counting. The hydrophilic PE species contained relatively much more 3 H label than those of PS, which indicates that they are more readily decarboxylated than the hydrophobic ones. Control experiments showed that differences in [ 3 H]PS and -PE molecular species profiles are not due to (i) incorporation of 3 H label to some PE species via alternative pathways, (ii) differences in degradation or remodeling among species, or (iii) selective decarboxylation of PS molecular species by the enzyme. Therefore, hydrophilic PS species are indeed decarboxylated faster than the hydrophobic ones. The rate of decarboxylation decreased systematically with hydrophobicity, strongly suggesting that formation of so called activated monomers, i.e. lipid molecules perpendicularly displaced from the membrane (Jones, J. D., and Thompson, T. E. (1990) Biochemistry 29, 1593-1600), is the rate-limiting step in the transport of PS from the endoplasmic reticulum to mitochondria. The formation of activated monomers and thus the rate of transfer is probably greatly enhanced by frequent collisions between the two membranes which tend to be closely associated. The present data also provides a feasible explanation why hydrophilic molecular species in these cells are much more abundant in PE as compared with PS, its immediate precursor.In many mammalian cells in culture most of phosphatidylethanolamine (PE) 1 is synthesized from phosphatidylserine (PS) in a process which involves transfer of PS from the endoplasmic reticulum (ER) to mitochondria, decarboxylation by PS decarboxylase (PSD) located in the inner mitochondrial membrane (1) and transfer of the nascent PE back to ER (2-7). Transport of PS to mitochondria (i) is ATP-dependent (8), (ii) does not seem to require soluble cytoplasmic components, including lipid transfer proteins (8, 9) or the cytoskeleton (8), (iii) takes place only between autologous ER membrane and mitochondria (10), and (iv) involves mainly newly made PS (11,12). Based on these facts, it has been suggested that the transport occurs upon ...
To study the translocation of phosphatidylserine (PS) from plasma membrane to mitochondria, dipyrene PS molecules (diPyr(n)PS; n=acyl chain length) were introduced to the plasma membrane of baby hamster kidney cells (BHK cells) using either cyclodextrin-mediated monomer transfer or fusion of cationic vesicles. Translocation of diPyr(n)PS to mitochondria was assessed based on decarboxylation by mitochondrial PS decarboxylase (PSD). It was found that the rate of translocation diminishes systematically with acyl chain length (molecular hydrophobicity) of diPyr(n)PS. Using an in vitro assay, it was shown that the spontaneous translocation rates of long-chain diPyr(n)PS species are similar to those of common natural PS species, thus supporting the biological relevance of the data. These results, and other data arguing against the involvement of vesicular traffic and lipid transfer proteins, imply that spontaneous monomeric diffusion via the cytoplasm is the main mechanism of PS movement from the plasma membrane to mitochondria. This finding could explain why a major fraction of PS synthesized by BHK cells consists of hydrophobic species: such species have little tendency to efflux from the plasma membrane to mitochondria where they would be decarboxylated. Thus, adequate molecular hydrophobicity seems to be crucial for the maintenance of high PS content in the inner leaflet of the plasma membrane.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.