Involvement of sphingosine 1-phosphate signaling in insulin-like growth factor-II/mannose 6-phosphate receptor trafficking from endosome to the trans-Golgi network.

The insulin-like growth factor II/mannose 6-phosphate (IGF-II/M6P) receptor is a multifunctional glycoprotein not only play roles in IGF-II degradation and pro-TGFß activation but binding to and transport M6P-bearing lysosomal enzymes from the trans-Golgi network (TGN) or the cell surface to lysosomes. At present, information regarding a retrograde transport of IGF-II/M6P receptor from endosomes to the TGN is still limited. We show here that a continuous ligand-dependent activation of sphingosine 1-phosphate receptor type 3 (S1P3R) on the endosomal membranes is required for subsequent recycling back of cargo-unloaded IGF-II/M6P receptors to the TGN. We have further clarified that Gq coupled with S1P3R plays a critical role in the activation of casein kinase 2, which phosphorylates and keeps PACS1 connector protein active for the association with IGF-II/M6P receptors, which enables transport carrier formation with the aid of other adaptor proteins toward the TGN. These findings shed light on the molecular mechanism underlying how continuous activation of the S1P receptor and subsequent downstream Gq signaling regulates the retrograde transport of the empty IGF-II/M6P receptors back to the TGN.

Arf1-dependent LRBA recruitment to Rab4 endosomes is required for endolysosome homeostasis.

Deleterious mutations in the lipopolysaccharide responsive beige-like anchor protein (LRBA) gene cause severe childhood immune dysregulation. The complexity of the symptoms involving multiple organs and the broad range of unpredictable clinical manifestations of LRBA deficiency complicate the choice of therapeutic interventions. Although LRBA has been linked to Rab11-dependent trafficking of the immune checkpoint protein CTLA-4, its precise cellular role remains elusive. We show that LRBA, however, only slightly colocalizes with Rab11. Instead, LRBA is recruited by members of the small GTPase Arf protein family to the TGN and to Rab4+ endosomes, where it controls intracellular traffic. In patient-derived fibroblasts, loss of LRBA led to defects in the endosomal pathway promoting the accumulation of enlarged endolysosomes and lysosome secretion. Thus, LRBA appears to regulate flow through the endosomal system on Rab4+ endosomes. Our data strongly suggest functions of LRBA beyond CTLA-4 trafficking and provide a conceptual framework to develop new therapies for LRBA deficiency.

The Rab6 post-Golgi secretory pathway contributes to herpes simplex virus 1 (HSV-1) egress.

Herpes simplex virus 1 (HSV-1) is an alpha herpesvirus that infects a majority of the world population. The mechanisms and cellular host factors involved in the intracellular transport and exocytosis of HSV-1 particles are not fully understood. To elucidate these late steps in the replication cycle, we developed a live-cell fluorescence microscopy assay of HSV-1 virion intracellular trafficking and exocytosis. This method allows us to track individual virus particles and identify the precise moment and location of particle exocytosis using a pH-sensitive reporter. We show that HSV-1 uses the host cell's post-Golgi secretory pathway during egress. The small GTPase, Rab6, binds to nascent secretory vesicles at the trans-Golgi network and plays important, but non-essential, roles in vesicle traffic and exocytosis at the plasma membrane, therefore making it a useful marker of the Golgi and post-Golgi secretory pathway. We show that HSV-1 particles colocalize with Rab6a in the region of the Golgi, cotraffic with Rab6a to the cell periphery, and undergo exocytosis from Rab6a vesicles. Consistent with previous reports, we find that HSV-1 particles accumulate at preferential egress sites in infected cells. The secretory pathway mediates this preferential/polarized egress, since Rab6a vesicles accumulate near the plasma membrane similarly in uninfected cells. These data suggest that, following particle envelopment, HSV-1 egress follows a pre-existing cellular secretory pathway to exit infected cells rather than novel, virus-induced mechanisms. IMPORTANCE: Herpes simplex virus 1 (HSV-1) infects a majority of people. It establishes a life-long latent infection and occasionally reactivates, typically causing characteristic oral or genital lesions. Rarely in healthy natural hosts, but more commonly in zoonotic infections and in elderly, newborn, or immunocompromised patients, HSV-1 can cause severe herpes encephalitis. The precise cellular mechanisms used by HSV-1 remain an important area of research. In particular, the egress pathways that newly assembled virus particles use to exit from infected cells are unclear. In this study, we used fluorescence microscopy to visualize individual virus particles exiting from cells and found that HSV-1 particles use the pre-existing cellular secretory pathway.

Consecutive palmitoylation and phosphorylation orchestrates NLRP3 membrane trafficking and inflammasome activation.

NLRP3 inflammasome activation, essential for cytokine secretion and pyroptosis in response to diverse stimuli, is closely associated with various diseases. Upon stimulation, NLRP3 undergoes subcellular membrane trafficking and conformational rearrangements, preparing itself for inflammasome assembly at the microtubule-organizing center (MTOC). Here, we elucidate an orchestrated mechanism underlying these ordered processes using human and murine cells. Specifically, NLRP3 undergoes palmitoylation at two sites by palmitoyl transferase zDHHC1, facilitating its trafficking between subcellular membranes, including the mitochondria, trans-Golgi network (TGN), and endosome. This dynamic trafficking culminates in the localization of NLRP3 to the MTOC, where LATS1/2, pre-recruited to MTOC during priming, phosphorylates NLRP3 to further facilitate its interaction with NIMA-related kinase 7 (NEK7), ultimately leading to full NLRP3 activation. Consistently, Zdhhc1-deficiency mitigated LPS-induced inflammation and conferred protection against mortality in mice. Altogether, our findings provide valuable insights into the regulation of NLRP3 membrane trafficking and inflammasome activation, governed by palmitoylation and phosphorylation events.

Intracellular Trafficking Defects in Congenital Intestinal and Hepatic Diseases.

Enterocytes and liver cells fulfill important metabolic and barrier functions and are responsible for crucial vectorial secretive and absorptive processes. To date, genetic diseases affecting metabolic enzymes or transmembrane transporters in the intestine and the liver are better comprehended than mutations affecting intracellular trafficking. In this review, we explore the emerging knowledge on intracellular trafficking defects and their clinical manifestations in both the intestine and the liver. We provide a detailed overview including more investigated diseases such as the canonical, variant and associated forms of microvillus inclusion disease, as well as recently described pathologies, highlighting the complexity and disease relevance of several trafficking pathways. We give examples of how intracellular trafficking hubs, such as the apical recycling endosome system, the trans-Golgi network, lysosomes, or the Golgi-to-endoplasmic reticulum transport are involved in the pathomechanism and lead to disease. Ultimately, understanding these processes could spark novel therapeutic approaches, which would greatly improve the quality of life of the affected patients.

Furin Egress from the TGN is Regulated by Membrane-Associated RING-CH Finger (MARCHF) Proteins and Ubiquitin-Specific Protease 32 (USP32) via Nondegradable K33-Polyubiquitination.

Furin primarily localizes to the trans-Golgi network (TGN), where it cleaves and activates a broad range of immature proproteins that play critical roles in cellular homeostasis, disease progression, and infection. Furin is retrieved from endosomes to the TGN after being phosphorylated, but it is still unclear how furin exits the TGN to initiate the post-Golgi trafficking and how its activity is regulated in the TGN. Here three membrane-associated RING-CH finger (MARCHF) proteins (2, 8, 9) are identified as furin E3 ubiquitin ligases, which catalyze furin K33-polyubiquitination. Polyubiquitination prevents furin from maturation by blocking its ectodomain cleavage inside cells but promotes its egress from the TGN and shedding. Further ubiquitin-specific protease 32 (USP32) is identified as the furin deubiquitinase in the TGN that counteracts the MARCHF inhibitory activity on furin. Thus, the furin post-Golgi trafficking is regulated by an interplay between polyubiquitination and phosphorylation. Polyubiquitination is required for furin anterograde transport but inhibits its proprotein convertase activity, and phosphorylation is required for furin retrograde transport to produce fully active furin inside cells.

Fatty acid synthesis promotes inflammasome activation through NLRP3 palmitoylation.

Despite its significance, the role of lipid metabolism in NLRP3 inflammasome remains elusive. Here, we reveal a critical role for fatty acid synthase (FASN) in NLRP3 inflammasome activation. We demonstrate that pharmacological or genetic depletion of FASN dampens NLRP3 activation in primary mouse and human macrophages and in mice. This disruption in NLRP3 activation is contingent upon FASN activity. Accordingly, abolishing cellular palmitoylation, a post-translational modification in which the FASN product palmitate is reversibly conjugated to cysteine residues of target proteins, blunts inflammasome signaling. Correspondingly, an acyl-biotin exchange assay corroborated NLRP3 palmitoylation. Mechanistically, Toll-like receptor (TLR) ligation introduces palmitoylation at NLRP3 Cys898, permitting NLRP3 translocation to dispersed trans-Golgi network (dTGN) vesicles, the site of inflammasome assembly, upon NLRP3 activation. Accordingly, the NLRP3 Cys898 mutant exhibits reduced palmitoylation, limited translocation to the dTGN compartment, and diminished inflammasome activation. These results underscore mechanistic insights through which lipid metabolism licenses NLRP3 inflammasome assembly and activation.

Localization of the MTP4 transporter to trans-Golgi network in pollen tubes of Arabidopsis thaliana.

Zinc (Zn) is an essential element for plants. Numerous proteins in different cellular compartments require Zn for their structure and function. Zn can be toxic when it accumulates in high levels in the cytoplasm. Therefore, Zn homeostasis at tissue, cell, and organelle levels is vital for plant growth. A part of the metal tolerance protein (MTP) / Cation Diffusion Facilitator (CDF) transporters functions as Zn transporters, exporting Zn from the cytosol to various membrane compartments. In Arabidopsis thaliana, MTP1, MTP2, MTP3, MTP4, MTP5, and MTP12 are classified as Zn transporters (Zn-CDF). In this study, we systematically analyzed the localization of GFP-fused Zn-CDFs in the leaf epidermal cells of Nicotiana benthamiana. As previously reported, MTP1 and MTP3 were localized to tonoplast, MTP2 to endoplasmic reticulum, and MTP5 to Golgi. In addition, we identified the localization of MTP4 to trans-Golgi Network (TGN). Since MTP4 is specifically expressed in pollen, we analyzed the localization of MTP4-GFP in the Arabidopsis pollen tubes and confirmed that it is in the TGN. We also showed the Zn transport capability of MTP4 in yeast cells. We then analyzed the phenotype of an mtp4 T-DNA insertion mutant under both limited and excess Zn conditions. We found that their growth and fertility were not largely different from the wild-type. Our study has paved the way for investigating the possible roles of MTP4 in metallating proteins in the secretory pathway or in exporting excess Zn through exocytosis. In addition, our system of GFP-fused MTPs will help study the mechanisms for targeting transporters to specific membrane compartments.

SNX32 Regulates Sorting and Trafficking of Activated EGFR to the Lysosomal Degradation Pathway.

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 lipid flippase ATP8A1 regulates the recruitment of ARF effectors to the trans-Golgi Network.

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.

EYA protein complex is required for Wntless retrograde trafficking from endosomes to Golgi.

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.

MARCH1 and MARCH2 inhibit pseudorabies virus replication by trapping the viral cell-to-cell fusion complex in 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.

MARCH8 inhibits pseudorabies virus replication by trapping the viral cell-to-cell fusion complex in the trans-Golgi network.

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 BICD2 dynein cargo adaptor binds to the HPV16 L2 capsid protein and promotes HPV infection.

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.

Golgi-associated retrograde protein (GARP) complex-dependent endosomes to trans Golgi network retrograde trafficking is controlled by Rab4b.

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. CONCLUSION: 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.

NLRP3 Cys126 palmitoylation by ZDHHC7 promotes inflammasome activation.

Nucleotide oligomerization domain (NOD)-like receptor protein 3 (NLRP3) inflammasome hyperactivation contributes to many human chronic inflammatory diseases, and understanding how NLRP3 inflammasome is regulated can provide strategies to treat inflammatory diseases. Here, we demonstrate that NLRP3 Cys126 is palmitoylated by zinc finger DHHC-type palmitoyl transferase 7 (ZDHHC7), which is critical for NLRP3-mediated inflammasome activation. Perturbing NLRP3 Cys126 palmitoylation by ZDHHC7 knockout, pharmacological inhibition, or modification site mutation diminishes NLRP3 activation in macrophages. Furthermore, Cys126 palmitoylation is vital for inflammasome activation in vivo. Mechanistically, ZDHHC7-mediated NLRP3 Cys126 palmitoylation promotes resting NLRP3 localizing on the trans-Golgi network (TGN) and activated NLRP3 on the dispersed TGN, which is indispensable for recruitment and oligomerization of the adaptor ASC (apoptosis-associated speck-like protein containing a CARD). The activation of NLRP3 by ZDHHC7 is different from the termination effect mediated by ZDHHC12, highlighting versatile regulatory roles of S-palmitoylation. Our study identifies an important regulatory mechanism of NLRP3 activation that suggests targeting ZDHHC7 or the NLRP3 Cys126 residue as a potential therapeutic strategy to treat NLRP3-related human disorders.

The role of the AP-1 adaptor complex in outgoing and incoming membrane traffic.

The AP-1 adaptor complex is found in all eukaryotes, but it has been implicated in different pathways in different organisms. To look directly at AP-1 function, we generated stably transduced HeLa cells coexpressing tagged AP-1 and various tagged membrane proteins. Live cell imaging showed that AP-1 is recruited onto tubular carriers trafficking from the Golgi apparatus to the plasma membrane, as well as onto transferrin-containing early/recycling endosomes. Analysis of single AP-1 vesicles showed that they are a heterogeneous population, which starts to sequester cargo 30 min after exit from the ER. Vesicle capture showed that AP-1 vesicles contain transmembrane proteins found at the TGN and early/recycling endosomes, as well as lysosomal hydrolases, but very little of the anterograde adaptor GGA2. Together, our results support a model in which AP-1 retrieves proteins from post-Golgi compartments back to the TGN, analogous to COPI's role in the early secretory pathway. We propose that this is the function of AP-1 in all eukaryotes.

Unveiling the TRAPP: The role of plant TRAPPII in adaptive growth decisions.

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.

Regulation of adaptive growth decisions via phosphorylation of the TRAPPII complex in Arabidopsis.

Plants often adapt to adverse or stress conditions via differential growth. The trans-Golgi network (TGN) has been implicated in stress responses, but it is not clear in what capacity it mediates adaptive growth decisions. In this study, we assess the role of the TGN in stress responses by exploring the previously identified interactome of the Transport Protein Particle II (TRAPPII) complex required for TGN structure and function. We identified physical and genetic interactions between AtTRAPPII and shaggy-like kinases (GSK3/AtSKs) and provided in vitro and in vivo evidence that the TRAPPII phosphostatus mediates adaptive responses to abiotic cues. AtSKs are multifunctional kinases that integrate a broad range of signals. Similarly, the AtTRAPPII interactome is vast and considerably enriched in signaling components. An AtSK-TRAPPII interaction would integrate all levels of cellular organization and instruct the TGN, a central and highly discriminate cellular hub, as to how to mobilize and allocate resources to optimize growth and survival under limiting or adverse conditions.

Polarized transport requires AP-1-mediated recruitment of KIF13A and KIF13B at the trans-Golgi.

Neurons are polarized cells that require accurate membrane trafficking to maintain distinct protein complements at dendritic and axonal membranes. The Kinesin-3 family members KIF13A and KIF13B are thought to mediate dendrite-selective transport, but the mechanism by which they are recruited to polarized vesicles and the differences in the specific trafficking role of each KIF13 have not been defined. We performed live-cell imaging in cultured hippocampal neurons and found that KIF13A is a dedicated dendrite-selective kinesin. KIF13B confers two different transport modes, dendrite- and axon-selective transport. Both KIF13s are maintained at the trans-Golgi network by interactions with the heterotetrameric adaptor protein complex AP-1. Interference with KIF13 binding to AP-1 resulted in disruptions to both dendrite- and axon-selective trafficking. We propose that AP-1 is the molecular link between the sorting of polarized cargoes into vesicles and the recruitment of kinesins that confer polarized transport.