9.A.63 The Retromer-dependent Vacuolar Protein Sorting (R-VPS) Family 

Endosomes are dynamic organelles that need to combine the ability to successfully deliver proteins and lipids to the lysosome, and recycle others to the Golgi or the plasma membrane. Retromer, which is implicated in retrieval of proteins from endosomes to the Golgi or the plasma membrane, can act on vacuoles. Recycling of the transmembrane receptor Vps10 from vacuoles requires the retromer, the dynamin-like Vps1, and the Rab7 GTPase Ypt7. Retromer and Vps1 leave the vacuole together with the cargo, whereas Ypt7 stays behind, in agreement with its regulatory function. Recycled cargo then accumulates in endosomes and later in the Golgi, implying consecutive sorting steps to the final destination. Thus, retromer and Vps1 are essential to maintain vacuole membrane organization. Taken together, and retromer cooperates with Vps1 and the Rab Ypt7 protein to clear the vacuole of selected membrane proteins (Arlt et al. 2015). The involvement of endosomal microdomains in sorting has been reviewed (Norris and Grant 2020).

Retromer is an evolutionarily conserved multimeric protein complex that mediates intracellular transport of various vesicular cargoes and functions in a wide variety of cellular processes including polarized trafficking, developmental signaling and lysosome biogenesis. Through its interaction with the Rab GTPases and their effectors, membrane lipids, molecular motors, the endocytic machinery and actin nucleation promoting factors, retromer regulates sorting and trafficking of transmembrane proteins from endosomes to the trans-Golgi network (TGN) and the plasma membrane (Liu 2016). Retromer contributes to a diverse set of developmental, physiological and pathological processes (Liu 2016). A constituent of the retromer complex is ANKRD50 (Kvainickas et al. 2016).  The endosomal trafficking of signaling membrane proteins, such as receptors, transporters and channels, is mediated by the retromer-mediated sorting machinery, composed of a cargo-selective vacuolar protein sorting trimer and a membrane-deforming subunit of sorting nexin proteins. The isoforms, sorting nexin 5 (SNX5) and SNX6, play distinctive regulatory roles in retrograde membrane trafficking.  Structural determinants specific for retromer protein sorting nexin 5 in regulating subcellular retrograde membrane traffickinghave been determined (Chen et al. 2023).

The retromer was first identified in genetic screens for genes involved in endosome-to-Golgi retrieval of Vps10p, the transmembrane sorting receptor for the vacuolar hydrolase carboxypeptidase Y (CPY) in the yeast Saccharomyces cerevisiae (Liu 2016). It is the endosomal coat complex that serves both cargo recognition and membrane deformation functions essential for sorting and trafficking of transmembrane proteins. It is a heteropentameric assembly composed of the Vps35-Vps29-Vps26 trimer and the Vps5p with Vps17p dimer, the latter being the yeast homolog of sorting nexin (SNX) family members that can bind and bend membranes. The core complex of retromer, the Vps35-Vps29-Vps26 subcomplex, is conserved across all eukaryotes. Although the core complex has been shown to interact with cargo proteins and was hence termed the cargo-selective complex (CSC), it was later found that the SNX components of retromer are also involved in cargo recognition. Cargo selectivity arises from defined combinations of the CSC with SNX family members in a given vesicular transport pathway. In yeast, the CSC and the SNX dimer forms a stable, biochemically isolatable complex, but in higher eukaryotes, the interaction between the CSC and SNXs is weaker and more transient. In mammals, the retromer interacts with many other proteins and is functionally and mechanistically diversified in mediating distinct membrane trafficking pathways in a variety of cellular processes (Liu 2016). Bcl-xES, a BH4- and BH2-containing antiapoptotic protein, delays Bax oligomer formation and binds Apaf-1, blocking procaspase-9 activation (Schmitt et al. 2004).

The retromer complex is a trafficking assembly composed of at least three proteins - Vps26, Vps29 and Vps35 - in Saccharomyces cerevisiae. Mammalian retromer sorts transmembrane proteins from the endosome to the trans-Golgi network (TGN), and plays a role in multiple trafficking pathways, including recycling to the plasma membrane and regulation of cell polarity (Follett et al. 2017). Retromer and its interacting proteins have been linked to familial forms of neurodegenerative diseases such as Alzheimer's (AD) and Parkinson's (PD). The transmembrane domains in target proteins modulate sorting of membrane proteins in Toxoplasma gondii (Karsten et al. 2004).

Newly synthesized glycosylphosphatidylinositol-anchored proteins having a very long chain ceramide lipid moiety cluster and sort into specialized endoplasmic reticulum exit sites that are distinct from those used by transmembrane proteins, and the chain length of the ceramide in the endoplasmic reticulum membrane is critical for this sorting selectivity (Rodriguez-Gallardo et al. 2020).

Retromer is an endosomal multi-protein complex that organizes the endocytic recycling of a vast range of integral membrane proteins. Jimenez-Orgaz et al. 2017 established an additional retromer function in controlling the activity and localization of the late endosomal small GTPase RAB7. 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. Lysosomal accumulation of hyperactivated RAB7 results in a striking loss of RAB7 mobility and depletion of the inactive RAB7 pool on endomembranes. 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 as well as autophagosome formation around damaged mitochondria during Parkin-mediated mitophagy (Jimenez-Orgaz et al. 2017).

The lysosome (or vacuole in yeast) receives membrane proteins from the secretory, endocytic, and macroautophagy/autophagy pathways. Some of these membrane proteins are recycled. The transmembrane autophagy protein Atg27 is recycled from the vacuole membrane using a 2-step recycling process (Suzuki and Emr 2018). First, the Snx4 complex recycles Atg27 from the vacuole to the endosome. Then, the retromer complex mediates endosome-to-Golgi retrograde transport. Thus, 2 distinct protein complexes facilitate the sequential retrograde trafficking for Atg27 (Suzuki and Emr 2018). Atg27 was the first physiological substrate for the vacuole-to-endosome retrograde trafficking pathway.

Retromer is required to maintain lysosomal amino acid signaling through mTORC1 across species (Kvainickas et al. 2019). Without retromer, amino acids do not stimulate mTORC1 translocation to the lysosomal membrane, which leads to a loss of mTORC1 activity and increased induction of autophagy. This 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 a role for retromer in the regulation of mTORC1 activity and longevity (Kvainickas et al. 2019).

Retromer (VPS26/VPS35/VPS29) is a highly conserved eukaryotic protein complex that localizes to endosomes to sort transmembrane protein cargoes into vesicles and elongated tubules. It mediates retrieval pathways from endosomes to the trans-Golgi network in all eukaryotes and further facilitates recycling pathways to the plasma membrane in metazoans. In cells, retromer engages multiple partners to orchestrate the formation of tubulovesicular structures, including sorting nexin (SNX) proteins, cargo adaptors, GTPases, regulators, and actin remodeling proteins. Retromer-mediated pathways are especially important for sorting cargoes required for neuronal maintenance, which links retromer loss or mutation to multiple human brain diseases and disorders. 3-d structures reveal retromer assembles into an arch-shaped scaffold and suggest the scaffold may be flexible and adaptable in cells. Interactions with cargo adaptors, particularly SNXs, likely orient the scaffold with respect to phosphatidylinositol-3-phosphate (PtdIns3P)-enriched membranes. Pharmacological small molecule chaperones stabilize retromer in cultured cell and mouse models. Chandra et al. 2020 reviewed structural and biophysical advances in understanding retromer structure.