Mammalian dynamin-like protein DLP1 tubulates membranes
Mammalian dynamin-like protein DLP1 tubulates membranes
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DOI：
10.1091/mbc.12.9.2894
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发表时间：
2001-09-01
影响因子：
3.3
通讯作者：
McNiven, MA
中科院分区：
文献类型：
--
作者：
Yoon, Y;Pitts, KR;McNiven, MA
动力蛋白是一种具有机械化学性质的大GTP酶，已知其可使膜收缩和形成平板。最近鉴定的哺乳动物动力蛋白样蛋白（DLP 1）是培养细胞中线粒体和内质网的适当细胞分布所必需的。在这项研究中，我们研究了DLP 1的能力，以重塑膜类似于传统的发动蛋白。我们发现，一个GTP酶缺陷的突变体，DLP 1-K38 A，在培养的细胞中的表达导致形成大的细胞质聚集体。表达DLP 1-K38 A的细胞的电子显微镜（EM）显示这些聚集体由直径一致的膜小管组成。高放大率的EM显示存在许多规则的条纹沿着个别膜小管，和免疫金标记证实了与这些结构的DLP 1的协会。使用重组DLP 1和标记的GTP的生化实验证明，DLP 1-K38 A结合但不水解或释放GTP。此外，与野生型DLP 1相比，DLP 1-K38 A对膜的亲和力增加。为了测试DLP 1是否可以在体外使膜成片状，将重组DLP 1与合成脂质体和核苷酸组合。我们发现，在鸟苷-5 ' -O-（3-硫代）三磷酸（GTP γ S）存在下，DLP 1蛋白单独组装成可沉积的大分子结构，但GTP不存在。GTP γ S处理的DLP 1的EM显示堆叠的螺旋环结构的簇。当脂质体包含在DLP 1中时，观察到形成与体内形成的那些尺寸相似的长膜小管。GTP γ S的加入极大地增强了膜小管的形成，表明GTP结合形式的DLP 1使脂质体变形为小管，就像DLP 1-K38 A在体内所做的那样。这些结果提供了动力蛋白家族成员DLP 1能够在活细胞和体外形成膜的第一个证据。此外，这些发现还表明，尽管与常规发动蛋白（35%）的同源性有限，但这些蛋白质以类似的方式重塑膜。
Dynamins are large GTPases with mechanochemical properties that axe known to constrict and tabulate membranes. A recently identified mammalian dynamin-like protein (DLP1) is essential for the proper cellular distribution of mitochondria and the endoplasmic reticulum in cultured cells. In this study, we investigated the ability of DLP1 to remodel membranes similar to conventional dynamin. We found that the expression of a GTPase-defective mutant, DLP1-K38A, in cultured cells led to the formation of large cytoplasmic aggregates. Electron microscopy (EM) of cells expressing DLP1-K38A revealed that these aggregates were comprised of membrane tubules of a consistent diameter. High-magnification EM revealed the presence of many regular striations along individual membrane tubules, and immunogold labeling confirmed the association of DLP1 with these structures. Biochemical experiments with the use of recombinant DLP1 and labeled GTP demonstrated that DLP1-K38A binds but does not hydrolyze or release GTP. Furthermore, the affinity of DLP1-K38A for membrane is increased compared with wild-type DLP1. To test whether DLP1 could tabulate membrane in vitro, recombinant DLP1 was combined with synthetic liposomes and nucleotides. We found that DLP1 protein alone assembled into sedimentable macromolecular structures in the presence of guanosine-5 ' -O-(3-thio)triphosphate (GTP gammaS) but not GTP. EM of the GTP gammaS-treated DLP1 revealed clusters of stacked helical ring structures. When liposomes were included with DLP1, formation of long membrane tubules similar in size to those formed in vivo was observed. Addition of GTP gammaS greatly enhanced membrane tubule formation, suggesting the GTP-bound form of DLP1 deforms liposomes into tubules as the DLP1-K38A does in vivo. These results provide the first evidence that the dynamin family member, DLP1, is able to tabulate membranes both in living cells and in vitro. Furthermore, these findings also indicate that despite the limited homology to conventional dynamins (35%) these proteins remodel membranes in a similar manner.