Department of Biochemistry and Molecular Biology, Monash University, Wellington Road, Clayton 3800, Australia.
J Cell Sci. 2010 Sep 15;123(Pt 18):3071-83. doi: 10.1242/jcs.060103. Epub 2010 Aug 24.
Phosphatidylinositol 3-phosphate [PtdIns(3)P] regulates endocytic trafficking and the sorting of receptors through early endosomes, including the rapid recycling of transferrin (Tfn). However, the phosphoinositide phosphatase that selectively opposes this function is unknown. The myotubularins are a family of eight catalytically active and six inactive enzymes that hydrolyse PtdIns(3)P to form PtdIns. However, the role each myotubularin family member plays in regulating endosomal PtdIns(3)P and thereby endocytic trafficking is not well established. Here, we identify the myotubularin family member MTMR4, which localizes to early endosomes and also to Rab11- and Sec15-positive recycling endosomes. In cells with MTMR4 knockdown, or following expression of the catalytically inactive MTMR4, MTMR4(C407A), the number of PtdIns(3)P-decorated endosomes significantly increased. MTMR4 overexpression delayed the exit of Tfn from early endosomes and its recycling to the plasma membrane. By contrast, expression of MTMR4(C407A), which acts as a dominant-negative construct, significantly accelerated Tfn recycling. However, in MTMR4 knockdown cells Tfn recycling was unchanged, suggesting that other MTMs might also contribute to recycling. MTMR4 regulated the subcellular distribution of Rab11 and, in cells with RNAi-mediated knockdown of MTMR4, Rab11 was directed away from the pericentriolar recycling compartment. The subcellular distribution of VAMP3, a v-SNARE protein that resides in recycling endosomes and endosome-derived transport vesicles, was also regulated by MTMR4. Therefore, MTMR4 localizes at the interface of early and recycling endosomes to regulate trafficking through this pathway.
磷脂酰肌醇 3-磷酸 [PtdIns(3)P] 通过早期内涵体调节内吞运输和受体分拣，包括转铁蛋白（Tfn）的快速再循环。然而，选择性拮抗此功能的磷酸肌醇磷酸酶尚不清楚。肌管素是一个由八个催化活性和六个非活性酶组成的家族，它们将 PtdIns(3)P 水解为 PtdIns。然而，每个肌管素家族成员在调节内体 PtdIns(3)P 从而调节内吞运输方面的作用尚未得到很好的确定。在这里，我们鉴定了肌管素家族成员 MTMR4，它定位于早期内涵体，也定位于 Rab11 和 Sec15 阳性的再循环内涵体。在 MTMR4 敲低的细胞中，或在表达催化失活的 MTMR4（MTMR4(C407A)）后，PtdIns(3)P 标记的内涵体数量显著增加。MTMR4 过表达延迟了 Tfn 从早期内涵体的出口及其再循环到质膜。相比之下，表达作为显性负构象的 MTMR4(C407A)，则显著加速了 Tfn 的再循环。然而，在 MTMR4 敲低的细胞中，Tfn 的再循环没有变化，这表明其他 MTMs 也可能有助于再循环。MTMR4 调节 Rab11 的亚细胞分布，在 MTMR4 的 RNAi 介导的敲低细胞中，Rab11 被引导远离中心体周围的再循环隔室。驻留在再循环内涵体和内涵体衍生的运输小泡中的 v-SNARE 蛋白 VAMP3 的亚细胞分布也受 MTMR4 调节。因此，MTMR4 定位于早期内涵体和再循环内涵体的界面，以调节通过该途径的运输。
