SNX32 is a member of the evolutionarily conserved Phox (PX) homology domain- and Bin/Amphiphysin/Rvs (BAR) domain- containing sorting nexin (SNX-BAR) family of proteins, which play important roles in sorting and membrane trafficking of endosomal cargoes. Although SNX32 shares the highest amino acid sequence homology with SNX6, and has been believed to function redundantly with SNX5 and SNX6 in retrieval of the cation-independent mannose-6-phosphate receptor (CI-MPR) from endosomes to the trans-Golgi network (TGN), its role(s) in intracellular protein trafficking remains largely unexplored. Here, we report that it functions in parallel with SNX1 in mediating epidermal growth factor (EGF)-stimulated postendocytic trafficking of the epidermal growth factor receptor (EGFR). Moreover, SNX32 interacts directly with EGFR, and recruits SNX5 to promote sorting of EGF-EGFR into multivesicular bodies (MVBs) for lysosomal degradation. Thus, SNX32 functions distinctively from other SNX-BAR proteins to mediate signaling-coupled endolysosomal trafficking of EGFR.

The membrane-associated RING-CH 8 protein (MARCH8), a member of the E3 ubiquitin ligase family, has broad-spectrum antiviral activity. However, some viruses hijack MARCH8 to promote virus replication, highlighting its dual role in the viral lifecycle. Most studies on MARCH8 have focused on RNA viruses, leaving its role in DNA viruses largely unexplored. Pseudorabies virus (PRV) is a large DNA virus that poses a potential threat to humans. In this study, we found that MARCH8 inhibited PRV replication at the cell-to-cell fusion stage. Interestingly, our findings proved that MARCH8 blocks gB cleavage by recruiting furin but this activity does not inhibit viral infection in vitro. Furthermore, we confirmed that MARCH8 inhibits cell-to-cell fusion independent of its E3 ubiquitin ligase activity but dependent on the interaction with the cell-to-cell fusion complex (gB, gD, gH, and gL). Finally, we discovered that the distribution of the cell-to-cell fusion complex is significantly altered and trapped within the trans-Golgi network. Overall, our results indicate that human MARCH8 acts as a potent antiviral host factor against PRV via trapping the cell-to-cell fusion complex in the trans-Golgi network.

The membrane-associated RING-CH (MARCH) family of proteins are members of the E3 ubiquitin ligase family and are essential for a variety of biological functions. Currently, MARCH proteins are discovered to execute antiviral functions by directly triggering viral protein degradation or blocking the furin cleavage of viral class I fusion proteins. Here, we report a novel antiviral mechanism of MARCH1 and MARCH2 (MARCH1/2) in the replication of Pseudorabies virus (PRV), a member of the Herpesviridae family. We discovered MARCH1/2 restrict PRV replication at the cell-to-cell fusion step. Furthermore, MARCH1/2 block gB cleavage, and this is dependent on their E3 ligase activity. Interestingly, the blocking of gB cleavage by MARCH1/2 does not contribute to their antiviral activity in vitro. We discovered that MARCH1/2 are associated with the cell-to-cell fusion complex of gB, gD, gH, and gL and trap these viral proteins in the trans-Golgi network (TGN) rather than degrading them. Overall, we conclude that MARCH1/2 inhibit PRV by trapping the viral cell-to-cell fusion complex in TGN.

Formation of transport vesicles requires the coordinate activity of the coating machinery that selects cargo into the nascent vesicle and the membrane bending machinery that imparts curvature to the forming bud. Vesicle coating at the trans-Golgi Network (TGN) involves AP1, GGA2 and clathrin, which are recruited to membranes by activated ARF GTPases. The ARF activation at the TGN is mediated by the BIG1 and BIG2 guanine nucleotide exchange factors (GEFs). Membrane deformation at the TGN has been shown to be mediated by lipid flippases, including ATP8A1, that moves phospholipids from the inner to the outer leaflet of the TGN membrane. We probed a possible coupling between the coating and deformation machineries by testing for an interaction between BIG1, BIG2 and ATP8A1, and by assessing whether such an interaction may influence coating efficiency. Herein, we document that BIG1 and BIG2 co-localize with ATP8A1 in both, static and highly mobile TGN elements, and that BIG1 and BIG2 bind ATP8A1. We show that the interaction involves the catalytic Sec7 domain of the GEFs and the cytosolic C-terminal tail of ATP8A1. Moreover, we report that the expression of ATP8A1, but not ATP8A1 lacking the GEF-binding cytosolic tail, increases the generation of activated ARFs at the TGN and increases the selective recruitment of AP1, GGA2 and clathrin to TGN membranes. This occurs without increasing BIG1 or BIG2 levels at the TGN, suggesting that the binding of the ATP8A1 flippase tail to the Sec7 domain of BIG1/BIG2 increases their catalytic activity. Our results support a model in which a flippase component of the deformation machinery impacts the activity of the GEF component of the coating machinery.

Retrograde transport of WLS (Wntless) from endosomes to trans-Golgi network (TGN) is required for efficient Wnt secretion during development. However, the molecular players connecting endosomes to TGN during WLS trafficking are limited. Here, we identified a role for Eyes Absent (EYA) proteins during retrograde trafficking of WLS to TGN in human cell lines. By using worm, fly, and zebrafish models, we found that the EYA-secretory carrier-associated membrane protein 3 (SCAMP3) axis is evolved in vertebrates. EYAs form a complex and interact with retromer on early endosomes. Retromer-bound EYA complex recruits SCAMP3 to endosomes, which is necessary for the fusion of WLS-containing endosomes to TGN. Loss of EYA complex or SCAMP3 leads to defective transport of WLS to TGN and failed Wnt secretion. EYA mutations found in patients with hearing loss form a dysfunctional EYA-retromer complex that fails to activate Wnt signaling. These findings identify the EYA complex as a component of retrograde trafficking of WLS from the endosome to TGN.

During entry, human papillomavirus (HPV) traffics from the endosome to the trans Golgi network (TGN) and Golgi and then the nucleus to cause infection. Although dynein is thought to play a role in HPV infection, how this host motor recruits the virus to support infection and which entry step(s) requires dynein are unclear. Here we show that the dynein cargo adaptor BICD2 binds to the HPV L2 capsid protein during entry, recruiting HPV to dynein for transport of the virus along the endosome-TGN/Golgi axis to promote infection. In the absence of BICD2 function, HPV accumulates in the endosome and TGN and infection is inhibited. Cell-based and in vitro binding studies identified a short segment near the C-terminus of L2 that can directly interact with BICD2. Our results reveal the molecular basis by which the dynein motor captures HPV to promote infection and identify this virus as a novel cargo of the BICD2 dynein adaptor.

The cation-independent mannose 6-phosphate receptors (CI-M6PR) bind newly synthesized mannose 6-phosphate (Man-6-P)-tagged enzymes in the Golgi and transport them to late endosomes/lysosomes, providing them with degradative functions. Following the cargo delivery, empty receptors are recycled via early/recycling endosomes back to the trans-Golgi network (TGN) retrogradely in a dynein-dependent motion. One of the most widely used methods for studying the retrograde trafficking of CI-M6PR involves employing the CD8α-CI-M6PR chimera. This chimera, comprising a CD8 ectodomain fused with the cytoplasmic tail of the CI-M6PR receptor, allows for labeling at the plasma membrane, followed by trafficking only in a retrograde direction. Previous studies utilizing the CD8α-CI-M6PR chimera have focused mainly on colocalization studies with various endocytic markers under steady-state conditions. This protocol extends the application of the CD8α-CI-M6PR chimera to live cell imaging, followed by a quantitative analysis of its motion towards the Golgi. Additionally, we present an approach to quantify parameters such as speed and track lengths associated with the motility of CD8α-CI-M6PR endosomes using the Fiji plugin TrackMate. Key features • This assay is adapted from the methodology by Prof. Matthew Seaman for studying the retrograde trafficking of CI-M6PR by expressing CD8α-CI-M6PR chimera in HeLa cells. • The experiments include live-cell imaging of surface-labeled CD8α-CI-M6PR molecules, followed by a chase in cells. • Allows the monitoring of real-time motion of CD8α-CI-M6PR endosomes and facilitates calculation of kinetic parameters associated with endosome trajectories, e.g., speed and distance (run lengths).

Clathrin assembles into honeycomb-like lattices at the plasma membrane but also on internal membranes, such as at the Golgi and tubular endosomes. Clathrin assemblies primarily regulate the intracellular trafficking of different cargoes, but clathrin also has non-endocytic functions in cell adhesion through interactions with specific integrins, contributes to intraluminal vesicle formation by forming flat bilayered coats on endosomes and even assembles on kinetochore k-fibers during mitosis. In this Cell Science at a Glance article and the accompanying poster, we review our current knowledge on the different types of canonical and non-canonical membrane-associated clathrin assemblies in mammalian cells, as observed by thin-section or platinum replica electron microscopy in various cell types, and discuss how the structural plasticity of clathrin contributes to its functional diversity.

The regulation of intracellular membrane traffic is coupled with the cell\'s need to respond to environmental stimuli, which ultimately is critical for different processes such as cell growth and development. In this issue, Wiese et al. (https://www.doi.org/10.1083/jcb.202311125) explore the role of the trans-Golgi network (TGN) in stress response, exposing its role in mediating adaptive growth decisions.

BACKGROUND: The trafficking of cargoes from endosomes to the trans-Golgi network requires numerous sequential and coordinated steps. Cargoes are sorted into endosomal-derived carriers that are transported, tethered, and fused to the trans-Golgi network. The tethering step requires several complexes, including the Golgi-associated retrograde protein complex, whose localization at the trans-Golgi network is determined by the activity of small GTPases of the Arl and Rab family. However, how the Golgi-associated retrograde protein complex recognizes the endosome-derived carriers that will fuse with the trans-Golgi network is still unknown.
METHODS: We studied the retrograde trafficking to the trans-Golgi network by using fluorescent cargoes in cells overexpressing Rab4b or after Rab4b knocked-down by small interfering RNA in combination with the downregulation of subunits of the Golgi-associated retrograde protein complex. We used immunofluorescence and image processing (Super Resolution Radial Fluctuation and 3D reconstruction) as well as biochemical approaches to characterize the consequences of these interventions on cargo carriers trafficking.
RESULTS: We reported that the VPS52 subunit of the Golgi-associated retrograde protein complex is an effector of Rab4b. We found that overexpression of wild type or active Rab4b increased early endosomal to trans-Golgi network retrograde trafficking of the cation-independent mannose-6-phosphate receptor in a Golgi-associated retrograde protein complex-dependent manner. Conversely, overexpression of an inactive Rab4b or Rab4b knockdown attenuated this trafficking. In the absence of Rab4b, the internalized cation-independent mannose 6 phosphate receptor did not have access to VPS52-labeled structures that look like endosomal subdomains and/or endosome-derived carriers, and whose subcellular distribution is Rab4b-independent. Consequently, the cation-independent mannose-6-phosphate receptor was blocked in early endosomes and no longer had access to the trans-Golgi network.
CONCLUSIONS: Our results support that Rab4b, by controlling the sorting of the cation-independent mannose-6-phosphate receptor towards VPS52 microdomains, confers a directional specificity for cargo carriers en route to the trans-Golgi network. Given the importance of the endocytic recycling in cell homeostasis, disruption of the Rab4b/Golgi-associated retrograde protein complex-dependent step could have serious consequences in pathologies.