Endosomal recycling of transmembrane proteins requires sequence-dependent recognition of motifs present within their intracellular cytosolic domains. In this study, we have reexamined the role of retromer in the sequence-dependent endosome-to–trans-Golgi network (TGN) transport of the cation-independent mannose 6-phosphate receptor (CI-MPR). Although the knockdown or knockout of retromer does not perturb CI-MPR transport, the targeting of the retromer-linked sorting nexin (SNX)–Bin, Amphiphysin, and Rvs (BAR) proteins leads to a pronounced defect in CI-MPR endosome-to-TGN transport. The retromer-linked SNX-BAR proteins comprise heterodimeric combinations of SNX1 or SNX2 with SNX5 or SNX6 and serve to regulate the biogenesis of tubular endosomal sorting profiles. We establish that SNX5 and SNX6 associate with the CI-MPR through recognition of a specific WLM endosome-to-TGN sorting motif. From validating the CI-MPR dependency of SNX1/2–SNX5/6 tubular profile formation, we provide a mechanism for coupling sequence-dependent cargo recognition with the biogenesis of tubular profiles required for endosome-to-TGN transport. Therefore, the data presented in this study reappraise retromer’s role in CI-MPR transport.

Multiple coronaviruses have emerged independently in the past 20 years that cause lethal human diseases. Although vaccine development targeting these viruses has been accelerated substantially, there remain patients requiring treatment who cannot be vaccinated or who experience breakthrough infections. Understanding the common host factors necessary for the life cycles of coronaviruses may reveal conserved therapeutic targets. Here, we used the known substrate specificities of mammalian protein kinases to deconvolute the sequence of phosphorylation events mediated by three host protein kinase families (SRPK, GSK-3, and CK1) that coordinately phosphorylate a cluster of serine and threonine residues in the viral N protein, which is required for viral replication. We also showed that loss or inhibition of SRPK1/2, which we propose initiates the N protein phosphorylation cascade, compromised the viral replication cycle. Because these phosphorylation sites are highly conserved across coronaviruses, inhibitors of these protein kinases not only may have therapeutic potential against COVID-19 but also may be broadly useful against coronavirus-mediated diseases.

The dynein-2 microtubule motor is the retrograde motor for intraflagellar transport. Mutations in dynein-2 components cause skeletal ciliopathies, notably Jeune syndrome. Dynein-2 contains a heterodimer of two non-identical intermediate chains, WDR34 and WDR60. Here, we use knockout cell lines to demonstrate that each intermediate chain has a distinct role in cilium function. Using quantitative proteomics, we show that WDR34 KO cells can assemble a dynein-2 motor complex that binds IFT proteins yet fails to extend an axoneme, indicating complex function is stalled. In contrast, WDR60 KO cells do extend axonemes but show reduced assembly of dynein-2 and binding to IFT proteins. Both proteins are required to maintain a functional transition zone and for efficient bidirectional intraflagellar transport. Our results indicate that the subunit asymmetry within the dynein-2 complex is matched with a functional asymmetry between the dynein-2 intermediate chains. Furthermore, this work reveals that loss of function of dynein-2 leads to defects in transition zone architecture, as well as intraflagellar transport.

Complex machinery is required to drive secretory cargo export from the endoplasmic reticulum (ER), which is an essential process in eukaryotic cells. In vertebrates, the MIA3 gene encodes two major forms of transport and Golgi organization protein 1 (TANGO1S and TANGO1L), which have previously been implicated in selective trafficking of procollagen. Using genome engineering of human cells, light microscopy, secretion assays, genomics and proteomics, we show that disruption of the longer form, TANGO1L, results in relatively minor defects in secretory pathway organization and function, including having limited impacts on procollagen secretion. In contrast, loss of both long and short forms results in major defects in cell organization and secretion. These include a failure to maintain the localization of ERGIC53 (also known as LMAN1) and SURF4 to the ER–Golgi intermediate compartment and dramatic changes to the ultrastructure of the ER–Golgi interface. Disruption of TANGO1 causes significant changes in early secretory pathway gene and protein expression, and impairs secretion not only of large proteins, but of all types of secretory cargo, including small soluble proteins. Our data support a general role for MIA3/TANGO1 in maintaining secretory pathway structure and function in vertebrate cells.

The retromer complex acts as a scaffold for endosomal protein complexes that sort integral membrane proteins to various cellular destinations. The retromer complex is a heterotrimer of VPS29, VPS35, and VPS26. Two of these paralogues, VPS26A and VPS26B, are expressed in humans. Retromer dysfunction is associated with neurodegenerative disease, and recently, three VPS26A mutations (p.K93E, p.M112V, and p.K297X) were discovered to be associated with atypical parkinsonism. Here, we apply quantitative proteomics to provide a detailed description of the retromer interactome. By establishing a comparative proteomic methodology, we identify how this interactome is perturbed in atypical parkinsonism-associated VPS26A mutants. In particular, we describe a selective defect in the association of VPS26A (p.K297X) with the SNX27 cargo adaptor. By showing how a retromer mutant leads to altered endosomal sorting of specific PDZ ligand–containing cargo proteins, we reveal a new mechanism for perturbed endosomal cargo sorting in atypical parkinsonism.