Voelker D R
Lord and Taylor Laboratory for Lung Biochemistry, National Jewish Center for Immunology and Respiratory Medicine, Denver, Colorado 80206.
J Biol Chem. 1993 Apr 5;268(10):7069-74.
The general properties of phosphatidylserine synthesis and translocation to the inner mitochondrial membrane which occur in both intact and permeabilized cells are now shown to occur in cells that are disrupted by shearing. In disrupted cells the synthesis of phosphatidylserine requires ATP and is blocked by preventing Ca2+ sequestration. The translocation of nascent phosphatidylserine to the mitochondria also requires ATP in disrupted cells. Using disrupted cells it is possible to test whether the translocation of nascent phosphatidylserine to the mitochondria is restricted to autologous mitochondrial acceptors or is possible with heterologous mitochondrial acceptors. To examine the nature of the transport process, a donor and acceptor population of cells were prepared. The donor cells were poisoned with hydroxylamine which irreversibly inactivates phosphatidylserine decarboxylase and will prevent the conversion of nascent phosphatidylserine to phosphatidylethanolamine. The acceptor cells were not treated with hydroxylamine so that any phosphatidylserine generated in the donor cell that reached the inner mitochondrial membrane of the acceptor cells would be decarboxylated to form phosphatidylethanolamine. Disrupted donor cells were pulse labeled with [3H]serine for 40 min to permit the synthesis of [3H] phosphatidylserine, and then further incorporation of radiolabel was arrested by chelating Ca2+. When the prelabeled disrupted donor cells were mixed with disrupted acceptor cells the latter failed to decarboxylate the nascent phosphatidylserine. In control experiments the addition of hydroxylamine-poisoned cells did not alter the transfer of nascent phosphatidylserine to autologous mitochondria present in unpoisoned cells. These results indicate that the ATP-dependent translocation of phosphatidylserine to the mitochondria is a process that is restricted to the autologous organelle.
现已表明，磷脂酰丝氨酸合成以及转运至线粒体内膜的一般特性，在完整细胞和透化细胞中都会发生，在通过剪切破坏的细胞中同样如此。在破坏的细胞中，磷脂酰丝氨酸的合成需要ATP，并且通过阻止Ca2+螯合而被阻断。新生磷脂酰丝氨酸转运至线粒体在破坏的细胞中也需要ATP。利用破坏的细胞，可以测试新生磷脂酰丝氨酸向线粒体的转运是否仅限于自体线粒体受体，或者与异源线粒体受体进行转运是否可行。为了研究转运过程的性质，制备了供体细胞和受体细胞群体。供体细胞用羟胺处理，羟胺会不可逆地使磷脂酰丝氨酸脱羧酶失活，并阻止新生磷脂酰丝氨酸转化为磷脂酰乙醇胺。受体细胞未用羟胺处理，这样供体细胞中产生的任何到达受体细胞线粒体内膜的磷脂酰丝氨酸都会脱羧形成磷脂酰乙醇胺。用[3H]丝氨酸对破坏的供体细胞进行40分钟的脉冲标记，以允许合成[3H]磷脂酰丝氨酸，然后通过螯合Ca2+阻止放射性标记的进一步掺入。当将预先标记的破坏供体细胞与破坏的受体细胞混合时，后者无法使新生磷脂酰丝氨酸脱羧。在对照实验中，添加经羟胺处理的细胞不会改变新生磷脂酰丝氨酸向未处理细胞中自体线粒体的转运。这些结果表明，磷脂酰丝氨酸向线粒体的ATP依赖性转运是一个仅限于自体细胞器的过程。
