Control of RAB7 activity and localization through the retromer-TBC1D5 complex enables RAB7-dependent mitophagy.

Retromer is an endosomal multi-protein complex that organizes the endocytic recycling of a vast range of integral membrane proteins. Here, we establish an additional retromer function in controlling the activity and localization of the late endosomal small GTPase RAB7. Surprisingly, we found that RAB7 not only decorates late endosomes or lysosomes, but is also present on the endoplasmic reticulum, trans-Golgi network, and mitochondrial membranes, a localization that is maintained by retromer and the retromer-associated RAB7-specific GAP TBC1D5. In the absence of either TBC1D5 or retromer, RAB7 activity state and localization are no longer controlled and hyperactivated RAB7 expands over the entire lysosomal domain. This lysosomal accumulation of hyperactivated RAB7 results in a striking loss of RAB7 mobility and overall depletion of the inactive RAB7 pool on endomembranes. Functionally, we establish that this control of RAB7 activity is not required for the recycling of retromer-dependent cargoes, but instead enables the correct sorting of the autophagy related transmembrane protein ATG9a and autophagosome formation around damaged mitochondria during Parkin-mediated mitophagy.

Retromer- and WASH-dependent sorting of nutrient transporters requires a multivalent interaction network with ANKRD50.

Retromer and the associated actin-polymerizing WASH complex are essential for the endocytic recycling of a wide range of integral membrane proteins. A hereditary Parkinson's-disease-causing point mutation (D620N) in the retromer subunit VPS35 perturbs retromer's association with the WASH complex and also with the uncharacterized protein ankyrin-repeat-domain-containing protein 50 (ANKRD50). Here, we firmly establish ANKRD50 as a new and essential component of the SNX27-retromer-WASH super complex. Depletion of ANKRD50 in HeLa or U2OS cells phenocopied the loss of endosome-to-cell-surface recycling of multiple transmembrane proteins seen upon suppression of SNX27, retromer or WASH-complex components. Mass-spectrometry-based quantification of the cell surface proteome of ANKRD50-depleted cells identified amino acid transporters of the SLC1A family, among them SLC1A4, as additional cargo molecules that depend on ANKRD50 and retromer for their endocytic recycling. Mechanistically, we show that ANKRD50 simultaneously engages multiple parts of the SNX27-retromer-WASH complex machinery in a direct and co-operative interaction network that is needed to efficiently recycle the nutrient transporters GLUT1 (also known as SLC2A1) and SLC1A4, and potentially many other surface proteins.

Retromer and TBC1D5 maintain late endosomal RAB7 domains to enable amino acid-induced mTORC1 signaling.

Retromer is an evolutionarily conserved multiprotein complex that orchestrates the endocytic recycling of integral membrane proteins. Here, we demonstrate that retromer is also required to maintain lysosomal amino acid signaling through mTORC1 across species. Without retromer, amino acids no longer stimulate mTORC1 translocation to the lysosomal membrane, which leads to a loss of mTORC1 activity and increased induction of autophagy. Mechanistically, we show that its effect on mTORC1 activity is not linked to retromer's role in the recycling of transmembrane proteins. Instead, retromer cooperates with the RAB7-GAP TBC1D5 to restrict late endosomal RAB7 into microdomains that are spatially separated from the amino acid-sensing domains. Upon loss of retromer, RAB7 expands into the ragulator-decorated amino acid-sensing domains and interferes with RAG-GTPase and mTORC1 recruitment. Depletion of retromer in Caenorhabditis elegans reduces mTORC1 signaling and extends the lifespan of the worms, confirming an evolutionarily conserved and unexpected role for retromer in the regulation of mTORC1 activity and longevity.

Cargo-selective SNX-BAR proteins mediate retromer trimer independent retrograde transport.

The retromer complex, which recycles the cation-independent mannose 6-phosphate receptor (CI-MPR) from endosomes to the trans-Golgi network (TGN), is thought to consist of a cargo-selective VPS26-VPS29-VPS35 trimer and a membrane-deforming subunit of sorting nexin (SNX)-Bin, Amphyphysin, and Rvs (BAR; SNX-BAR) proteins. In this study, we demonstrate that heterodimers of the SNX-BAR proteins, SNX1, SNX2, SNX5, and SNX6, are the cargo-selective elements that mediate the retrograde transport of CI-MPR from endosomes to the TGN independently of the core retromer trimer. Using quantitative proteomics, we also identify the IGF1R, among more potential cargo, as another SNX5 and SNX6 binding receptor that recycles through SNX-BAR heterodimers, but not via the retromer trimer, in a ligand- and activation-dependent manner. Overall, our data redefine the mechanics of retromer-based sorting and call into question whether retromer indeed functions as a complex of SNX-BAR proteins and the VPS26-VPS29-VPS35 trimer.

Suppression of APC/CCdh1 has subtype specific biological effects in acute myeloid leukemia.

The E3 ubiquitin ligase and tumor suppressor APC/CCdh1 is crucial for cell cycle progression, development and differentiation in many cell types. However, little is known about the role of Cdh1 in hematopoiesis. Here we analyzed Cdh1 expression and function in malignant hematopoiesis. We found a significant decrease of Cdh1 in primary acute myeloid leukemia (AML) blasts compared to normal CD34+ cells. Thus, according to its important role in connecting cell cycle exit and differentiation, decreased expression of Cdh1 may be a mechanism contributing to the differentiation block in leukemogenesis. Indeed, knockdown (kd) of Cdh1 in HL-60 cell line (AML with maturation, FAB M2) led to less differentiated cells and a delay in PMA-induced differentiation. Acute promyelocytic leukemia (APL, FAB M3) is an AML subtype which is highly vulnerable to differentiation therapy with all-trans retinoic acid (ATRA). Accordingly, we found that APL is resistant to a Cdh1-kd mediated differentiation block. However, further depletion of Cdh1 in APL significantly reduced viability of leukemia cells upon ATRA-induced differentiation. Thus, low Cdh1 expression may be important in AML biology by contributing to the differentiation block and response to therapy depending on differences in the microenvironment and the additional genetic background.