The myotubularin phosphatase MTMR4 regulates sorting from early endosomes

Abstract

SummaryPhosphatidylinositol 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, MTMR4C407A , 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 endosomederived transport vesicles, was also regulated by MTMR4. Therefore, MTMR4 localizes at the interface of early and recycling endosomes to regulate trafficking through this pathway.

A phosphoinositide conversion mechanism for exit from endosomes

“…In addition to MTM1 depletion, loss of the endosomal PI(3)P-specific 3-phosphatases MTMR2 or MTMR4 has been shown to elevate cellular PI(3)P levels (Cao, Backer et al 2008;Naughtin, Sheffield et al 2010). Thus, other myotubularins might compensate for loss of MTM1 as was proposed previously (Laporte, Liaubet et al 2002).…”

“…While recycling remains unperturbed upon loss of MTMR4, over-expressed MTMR4 impairs TfR recycling and EGFR degradation via a mechanism that requires its phosphatase activity. Also, MTMR4 colocalizes with the exocyst subunit Sec15 and affects localization of VAMP3-containing endosomes (Lorenzo, Urbe et al 2006;Naughtin, Sheffield et al 2010). Additionally to PI(3)P, MTMR4 dephosphorylates RSmads at early endosomes and thereby negatively regulates TGF-β (transforming growth factor) (Yu, Pan et al 2010) and BMP (bone morphogenetic protein) signaling pathways (Yu, He et al 2013).…”

“…A similar, although less severe phenotype was seen upon depletion of the closely related PI 3-phosphatase MTMR1, an enzyme of so far unknown function, while loss of MTMR2 (a PI(3)P and PI(3,5)P 2 3-phosphatase, localized to early and late endosomes and implicated in degradative sorting (Cao, Backer et al 2008)), MTMR4 (a PI(3)P 3-phosphatase, localized to early and recycling endosomes and implicated in endosomal sorting (Naughtin, Sheffield et al 2010)) or MTMR7 (a PI(3)P and I(1,3)P 2 3-phospahatse, associated with Golgi-like structures (Mochizuki and Majerus 2003)) had no effect on TfR distribution or exocytosis (Figure 15). One might speculate that differences in tissue-specific and developmentally regulated expression patterns account for the need of MTM1 and MTMR1 in vivo.…”

“…In accordance with our data, TfR recycling remains unperturbed upon loss of MTMR4. However, sorting of TfR from early to recycling endosomes is impaired while over-expression of active MTMR4 has dominant negative effects on TfR recycling (Naughtin, Sheffield et al 2010). …”

“…Accordingly, MTMR2 could mediate degradative sorting or recycling from PI(3,5)P 2 -positive late endosomes (as discussed in 4.3.1). MTMR4, on the other hand, was shown to predominantly localize to early endosomes, but its depletion did not alter the overall recycling kinetics of TfR (Naughtin, Sheffield et al 2010). However, prior to monitoring exocytosis, we load the endosomal system with internalized ligand until saturation is reached.…”