IDENTIFICATION OF A LYSOSOMAL PROTEIN CAUSING LIPID TRANSFER, USING A FLUORESCENCE ASSAY DESIGNED TO MONITOR MEMBRANE-FUSION BETWEEN RAT-LIVER ENDOSOMES AND LYSOSOMES
IDENTIFICATION OF A LYSOSOMAL PROTEIN CAUSING LIPID TRANSFER, USING A FLUORESCENCE ASSAY DESIGNED TO MONITOR MEMBRANE-FUSION BETWEEN RAT-LIVER ENDOSOMES AND LYSOSOMES
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DOI：
10.1042/bj3080937
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发表时间：
1995-06-15
影响因子：
4.1
通讯作者：
LUZIO, JP
中科院分区：
文献类型：
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作者：
KUWANA, T;MULLOCK, BM;LUZIO, JP
在当前和先前的研究中[Mullock，Perez，库瓦纳，Gray和Luzio（1994）细胞生物学杂志126，1273-1182]，我们尝试使用基于稀释自猝灭荧光脂质探针十八烷基罗丹明的测定来研究内体-溶酶体融合。虽然荧光去猝灭的一些特征与在其他无细胞测定中观察到的特征一致，但我们现在已经证明荧光增加是由于溶酶体内脂质转移蛋白的泄漏。该蛋白被纯化，并发现是一个22 kDa的分子，序列，免疫学和功能特性强烈表明它是人G（M2）-激活蛋白的大鼠同源物。无论是22 kDa的蛋白质和重组人G（M2）-激活蛋白引起荧光去猝灭时，与十八烷基罗丹明加载的内体和溶酶体膜混合，或在脂质体系统。这些数据与G（M2）-激活蛋白作为十八烷基罗丹明转移蛋白一致。加入到无细胞内体-溶酶体含量混合测定中的22 kDa蛋白的抗体没有影响，尽管它们可以抑制由蛋白引起的荧光去猝灭。因此，该蛋白质在参与将配体从内体递送至溶酶体的任何融合事件中是不需要的。能够作为十八烷基罗丹明转移蛋白的蛋白质的细胞内细胞器内的存在表明需要谨慎的解释使用哺乳动物亚细胞组分的荧光去猝灭测定。
In the present and previous studies [Mullock, Perez, Kuwana, Gray and Luzio (1994) J. Cell Biol. 126, 1273-1182], we have attempted to investigate endosom-lysosome fusion using an assay based on the dilution of the self-quenching fluorescent lipid probe octadecylrhodamine. Although some characteristics of fluorescence dequenching were consistent with those observed in other cell-free assays, we have now demonstrated that increased fluorescence was due to leakage of an intralysosomal lipid-transfer protein. This protein was purified and found to be a 22 kDa molecule with sequence, immunological and functional characteristics strongly suggesting that it is the rat homologue of human G(M2)-activator protein. Both the 22 kDa protein and recombinant human G(M2)-activator protein caused fluorescence dequenching either when mixed with octadecylrhodamine-loaded endosomes and lysosomal membranes or in a liposome system. The data were consistent with G(M2)-activator protein acting as an octadecylrhodamine-transfer protein. Antibodies to the 22 kDa protein added to cell-free endosome-lysosome content-mixing assays had no effect, although they could inhibit fluorescence dequenching caused by the protein. Thus this protein is not required in any fusion event involved in delivery of ligands from endosomes to lysosomes. The existence within an intracellular organelle of a protein capable of acting as an octadecylrhodamine-transfer protein suggests the need for caution in the interpretation of fluorescence-dequenching assays using mammalian subcellular fractions.