Trafficking Function Research Articles

The TRAPP complexes: discriminating GTPases in context.

Correct localization of Rab GTPases in cells is critical for proper function in membrane trafficking. Guanine-nucleotide exchange factors (GEFs) act as the primary determinants of Rab localization by activating and stabilizing their Rab substrates on specific organelle and vesicle membranes. The TRAPP complexes TRAPPII and TRAPPIII are two related GEFs that use the same catalytic site to activate distinct Rabs, Rab11 and Rab1, respectively. The Rab C-terminal hypervariable domain (HVD) is an important specificity determinant for the budding yeast TRAPP complexes, with the length of the HVD playing a critical role in counter-selection. Several recent studies have used cryo-EM to illuminate how the yeast and metazoan TRAPP complexes identify and activate their substrates. This review summarizes recently characterized Rab substrate selection mechanisms and highlights how the membrane surface provides critical context for the GEF-GTPase interactions.

Assembly and stability of IFT-B complex and its function in BBSome trafficking.

The machinery of intraflagellar transport (IFT) consists of IFT motors and the ciliary cargo adaptors including IFT-A and IFT-B complexes and BBSome. IFT-B, which is composed of IFT-B1 and IFT-B2 subcomplexes, interacts with IFT motors and IFT-A during anterograde IFT and IFT-A during retrograde IFT while it is also implicated in BBSome trafficking. However, the assembly and stability of IFT-B and its regulation of anterograde IFT and BBSome trafficking remain not clear. Here, we show that IFT38 functions in the regulation of anterograde IFT and retrograde trafficking of BBSome. Deletion of IFT-B1 or IFT-B2 subunits results in differential instability of IFT-B1 and IFT-B2. The stability of IFT-B1 and IFT-B2 is mutually dependent and mediated by the connecting tetramer IFT38/5788/52. The formation of an intact IFT-B1 and IFT-B2 is not altered by the deletion of IFT38 of IFT-B2 and IFT52 of IFT-B1, respectively. Further analysis suggests a modular pathway for IFT-B assembly.

Nephrotic Syndrome Gene TBC1D8B Is Required for Endosomal Maturation and Nephrin Endocytosis in Drosophila.

Variants in TBC1D8B cause nephrotic syndrome. TBC1D8B is a GTPase-activating protein for Rab11 (RAB11-GAP) that interacts with nephrin, but how it controls nephrin trafficking or other podocyte functions remains unclear. We generated a stable deletion in Tbc1d8b and used microhomology-mediated end-joining for genome editing. Ex vivo functional assays utilized slit diaphragms in podocyte-like Drosophila nephrocytes. Manipulation of endocytic regulators and transgenesis of murine Tbc1d8b provided a comprehensive functional analysis of Tbc1d8b. A null allele of Drosophila TBC1D8B exhibited a nephrocyte-restricted phenotype of nephrin mislocalization, similar to patients with isolated nephrotic syndrome who have variants in the gene. The protein was required for rapid nephrin turnover in nephrocytes and for endocytosis of nephrin induced by excessive Rab5 activity. The protein expressed from the Tbc1d8b locus bearing the edited tag predominantly localized to mature early and late endosomes. Tbc1d8b was required for endocytic cargo processing and degradation. Silencing Hrs, a regulator of endosomal maturation, phenocopied loss of Tbc1d8b. Low-level expression of murine TBC1D8B rescued loss of the Drosophila gene, indicating evolutionary conservation. Excessive murine TBC1D8B selectively disturbed nephrin dynamics. Finally, we discovered four novel TBC1D8B variants within a cohort of 363 patients with FSGS and validated a functional effect of two variants in Drosophila, suggesting a personalized platform for TBC1D8B-associated FSGS. Variants in TBC1D8B are not infrequent among patients with FSGS. TBC1D8B, functioning in endosomal maturation and degradation, is essential for nephrin trafficking.

Molecular interplays of the Entamoeba histolytica endosomal sorting complexes required for transport during phagocytosis.

Entamoeba histolytica, the causative agent of human amoebiasis, exhibits a continuous membrane remodelling to exert its virulence properties. During this dynamic process, the Endosomal Sorting Complexes Required for Transport (ESCRT) machinery is a key player, particularly in phagocytosis, a virulence hallmark of this parasite. In addition to ESCRT, other molecules contribute to membrane remodelling, including the EhADH adhesin, EhRabs, actin, and the lysobisphosphatidic acid (LBPA). The endocytosis of a prey or molecules induces membrane invaginations, resulting in endosome and multivesicular bodies (MVBs) formation for cargo delivery into lysosomes. Alternatively, some proteins are recycled or secreted. Most of these pathways have been broadly characterized in other biological systems, but poorly described in protozoan parasites. Here, we encompass 10 years of ESCRT research in E. histolytica, highlighting the role of the ESCRT-I and ESCRT-III components and the EhADH and EhVps4-ATPase accessory proteins during phagocytosis. In particular, EhADH exhibits a multifunctional role along the endocytic pathway, from cargo recognition to endosome maturation and lysosomal degradation. Interestingly, the interaction of EhADH with EhVps32 seems to shape a concurrent route to the conventional one for MVBs biogenesis, that could optimize their formation. Furthermore, this adhesin is secreted, but its role in this event remains under study. Other components from the endosomal pathway, such as EhVps23 and LBPA, are also secreted. A proteomic approach performed here, using an anti-LBPA antibody, revealed that some proteins related to membrane trafficking, cellular transport, cytoskeleton dynamics, and transcriptional and translational functions are secreted and associated to LBPA. Altogether, the accumulated knowledge around the ESCRT machinery in E. histolytica, points it out as a dynamic platform facilitating the interaction of molecules participating in different cellular events. Seen as an integrated system, ESCRTs lead to a better understanding of E. histolytica phagocytosis.

Vitamin D3 alters macrophage phenotype and endosomal trafficking markers in dairy cattle naturally infected with Mycobacterium avium subsp. paratuberculosis.

Macrophages are important host defense cells in ruminant paratuberculosis (Johne’s Disease; JD), a chronic enteritis caused by Mycobacterium avium subsp. paratuberculosis (MAP). Classical macrophage functions of pathogen trafficking, degradation, and antigen presentation are interrupted in mycobacterial infection. Immunologic stimulation by 25-hydroxyvitamin D3 (25(OH)D3) and 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) enhances bovine macrophage function. The present study aimed to investigate the role of vitamin D3 on macrophage phenotype and endosomal trafficking of MAP in monocyte-derived macrophages (MDMs) cultured from JD-, JD+ subclinical, and JD+ clinically infected cattle. MDMs were pre-treated 100 ng/ml 25(OH)D3 or 4 ng/ml 1,25(OH)2D3 and incubated 24 hrs with MAP at 10:1 multiplicity of infection (MOI). In vitro MAP infection upregulated pro-inflammatory (M1) CD80 and downregulated resolution/repair (M2) CD163. Vitamin D3 generally decreased CD80 and increased CD163 expression. Furthermore, early endosomal marker Rab5 was upregulated 140× across all stages of paratuberculosis infection following in vitro MAP infection; however, Rab5 was reduced in MAP-activated MDMs from JD+ subclinical and JD+ clinical cows compared to healthy controls. Rab7 expression decreased in control and clinical cows following MDM infection with MAP. Both forms of vitamin D3 reduced Rab5 expression in infected MDMs from JD- control cows, while 1,25(OH)2D3 decreased Rab7 expression in JD- and JD+ subclinical animals regardless of MAP infection in vitro. Vitamin D3 promoted phagocytosis in MDMs from JD- and JD+ clinical cows treated with either vitamin D3 analog. Results from this study show exogenous vitamin D3 influences macrophage M1/M2 polarization and Rab GTPase expression within MDM culture.

Glucose metabolism and glycosylation link the gut microbiota to autoimmune diseases.

Carbohydrates serve as important energy sources and structural substances for human body as well as for gut microbes. As evidenced by the advances in immunometabolism, glucose metabolism and adenosine triphosphate (ATP) generation are deeply involved in immune cell activation, proliferation, and signaling transduction as well as trafficking and effector functions, thus contributing to immune response programming and assisting in host adaption to microenvironment changes. Increased glucose uptake, aberrant expression of glucose transporter 1 (e.g., GLU1), and abnormal glycosylation patterns have been identified in autoimmunity and are suggested as partially responsible for the dysregulated immune response and the modification of gut microbiome composition in the autoimmune pathogenesis. The interaction between gut microbiota and host carbohydrate metabolism is complex and bidirectional. Their impact on host immune homeostasis and the development of autoimmune diseases remains to be elucidated. This review summarized the current knowledge on the crosstalk of glucose metabolism and glycosylation in the host with intestinal microbiota and discussed their possible role in the development and progression of autoimmune diseases. Potential therapeutic strategies targeting glucose metabolism and glycosylation in modulating gut ecosystem and treating autoimmune diseases were discussed as well.

ER as master regulator of membrane trafficking and organelle function.

The endoplasmic reticulum (ER), which occupies a large portion of the cytoplasm, is the cell's main site for the biosynthesis of lipids and carbohydrate conjugates, and it is essential for folding, assembly, and biosynthetic transport of secreted proteins and integral membrane proteins. The discovery of abundant membrane contact sites (MCSs) between the ER and other membrane compartments has revealed that, in addition to its biosynthetic and secretory functions, the ER plays key roles in the regulation of organelle dynamics and functions. In this review, we will discuss how the ER regulates endosomes, lysosomes, autophagosomes, mitochondria, peroxisomes, and the Golgi apparatus via MCSs. Such regulation occurs via lipid and Ca2+ transfer and also via control of in trans dephosphorylation reactions and organelle motility, positioning, fusion, and fission. The diverse controls of other organelles via MCSs manifest the ER as master regulator of organelle biology.

Sponge/DOCK-dependent regulation of F-actin networks directing cortical cap behaviors and syncytial furrow ingression

In the early syncytial Drosophila embryo, rapid changes in filamentous actin networks and membrane trafficking pathways drive the formation and remodeling of cortical and furrow morphologies. Interestingly, genomic integrity and the completion of mitoses during cell cycles 10–13 depends on the formation of transient membrane furrows that serve to separate and anchor individual spindles during division. While substantial work has led to a better understanding of the core network components that are responsible for the formation of these furrows, less is known about the regulation that controls cytoskeletal and trafficking function. The DOCK protein Sponge was one of the first proteins identified as being required for syncytial furrow formation, and disruption of Sponge deeply compromises F-actin populations in the early embryo, but how this occurs is less clear. Here, we perform quantitative analysis of the effects of Sponge disruption on cortical cap growth, furrow formation, membrane trafficking, and cytoskeletal network regulation through live-imaging of the syncytial embryo. We find that membrane trafficking is relatively unaffected by the defects in branched actin networks that occur after Sponge disruption, but that Sponge acts as a master regulator of a diverse cohort of Arp2/3 regulatory proteins. As DOCK family proteins have been implicated in regulating GTP exchange on small GTPases, we also suggest that Rac GTPase activity bridges Sponge regulation to the regulators of Arp2/3 function. Finally, we demonstrate the phasic requirements for branched F-actin and linear F-actin networks in potentiating furrow ingression. In total, these results provide quantitative insights into how a large DOCK scaffolding protein coordinates the activity of a variety of different actin regulatory proteins to direct the remodeling of the apical cortex into cytokinetic-like furrows.

Inflammatory Cytokines Rewire the Proinsulin Interaction Network in Human Islets.

Aberrant biosynthesis and secretion of the insulin precursor proinsulin occurs in both type I and type II diabetes. Inflammatory cytokines are implicated in pancreatic islet stress and dysfunction in both forms of diabetes, but the mechanisms remain unclear. We sought to determine the effect of the diabetes-associated cytokines on proinsulin folding, trafficking, secretion, and β-cell function. Human islets were treated with interleukin-1β and interferon-γ for 48 hours, followed by analysis of interleukin-6, nitrite, proinsulin and insulin release, RNA sequencing, and unbiased profiling of the proinsulin interactome by affinity purification-mass spectrometry. Cytokine treatment induced secretion of interleukin-6, nitrites, and insulin, as well as aberrant release of proinsulin. RNA sequencing showed that cytokines upregulated genes involved in endoplasmic reticulum stress, and, consistent with this, affinity purification-mass spectrometry revealed cytokine induced proinsulin binding to multiple endoplasmic reticulum chaperones and oxidoreductases. Moreover, increased binding to the chaperone immunoglobulin binding protein was required to maintain proper proinsulin folding in the inflammatory environment. Cytokines also regulated novel interactions between proinsulin and type 1 and type 2 diabetes genome-wide association studies candidate proteins not previously known to interact with proinsulin (eg, Ataxin-2). Finally, cytokines induced proinsulin interactions with a cluster of microtubule motor proteins and chemical destabilization of microtubules with Nocodazole exacerbated cytokine induced proinsulin secretion. Together, the data shed new light on mechanisms by which diabetes-associated cytokines dysregulate β-cell function. For the first time, we show that even short-term exposure to an inflammatory environment reshapes proinsulin interactions with critical chaperones and regulators of the secretory pathway.

The Polarized Redistribution of the Contractile Vacuole to the Rear of the Cell is Critical for Streaming and is Regulated by PI(4,5)P2-Mediated Exocytosis

Dictyostelium discoideum amoebae align in a head to tail manner during the process of streaming during fruiting body formation. The chemoattractant cAMP is the chemoattractant regulating cell migration during this process and is released from the rear of cells. The process by which this cAMP release occurs has eluded investigators for many decades, but new findings suggest that this release can occur through expulsion during contractile vacuole (CV) ejection. The CV is an organelle that performs several functions inside the cell including the regulation of osmolarity, and discharges its content via exocytosis. The CV localizes to the rear of the cell and appears to be part of the polarity network, with the localization under the influence of the plasma membrane (PM) lipids, including the phosphoinositides (PIs), among those is PI(4,5)P2, the most abundant PI on the PM. Research on D. discoideum and neutrophils have shown that PI(4,5)P2 is enriched at the rear of migrating cells. In several systems, it has been shown that the essential regulator of exocytosis is through the exocyst complex, mediated in part by PI(4,5)P2-binding. This review features the role of the CV complex in D. discoideum signaling with a focus on the role of PI(4,5)P2 in regulating CV exocytosis and localization. Many of the regulators of these processes are conserved during evolution, so the mechanisms controlling exocytosis and membrane trafficking in D. discoideum and mammalian cells will be discussed, highlighting their important functions in membrane trafficking and signaling in health and disease.

The Puzzle of Hereditary Spastic Paraplegia: From Epidemiology to Treatment.

Inherited neurodegenerative pathology characterized by lower muscle tone and increasing spasticity in the lower limbs is termed hereditary spastic paraplegia (HSP). HSP is associated with changes in about 80 genes and their products involved in various biochemical pathways, such as lipid droplet formation, endoplasmic reticulum shaping, axon transport, endosome trafficking, and mitochondrial function. With the inheritance patterns of autosomal dominant, autosomal recessive, X-linked recessive, and mitochondrial inheritance, HSP is prevalent around the globe at a rate of 1–5 cases in every 100,000 individuals. Recent technology and medical interventions somewhat aid in recognizing and managing the malaise. However, HSP still lacks an appropriate and adequate therapeutic approach. Current therapies are based on the clinical manifestations observed in the patients, for example, smoothing the relaxant spastic muscle and physiotherapies. The limited clinical trial studies contribute to the absence of specific pharmaceuticals for HSPs. Our current work briefly explains the causative genes, epidemiology, underlying mechanism, and the management approach undertaken to date. We have also mentioned the latest approved drugs to summarise the available knowledge on therapeutic strategies for HSP.

The PH Domain and C-Terminal polyD Motif of Phafin2 Exhibit a Unique Concurrence in Animals.

Phafin2, a member of the Phafin family of proteins, contributes to a plethora of cellular activities including autophagy, endosomal cargo transportation, and macropinocytosis. The PH and FYVE domains of Phafin2 play key roles in membrane binding, whereas the C-terminal poly aspartic acid (polyD) motif specifically autoinhibits the PH domain binding to the membrane phosphatidylinositol 3-phosphate (PtdIns3P). Since the Phafin2 FYVE domain also binds PtdIns3P, the role of the polyD motif remains unclear. In this study, bioinformatics tools and resources were employed to determine the concurrence of the PH-FYVE module with the polyD motif among Phafin2 and PH-, FYVE-, or polyD-containing proteins from bacteria to humans. FYVE was found to be an ancient domain of Phafin2 and is related to proteins that are present in both prokaryotes and eukaryotes. Interestingly, the polyD motif only evolved in Phafin2 and PH- or both PH-FYVE-containing proteins in animals. PolyD motifs are absent in PH domain-free FYVE-containing proteins, which usually display cellular trafficking or autophagic functions. Moreover, the prediction of the Phafin2-interacting network indicates that Phafin2 primarily cross-talks with proteins involved in autophagy, protein trafficking, and neuronal function. Taken together, the concurrence of the polyD motif with the PH domain may be associated with complex cellular functions that evolved specifically in animals.

Ferritin Iron Responsive Elements (IREs) mRNA Interacts with eIF4G and Activates In Vitro Translation

Eukaryotic initiation factor (eIF) 4G plays an important role in assembling the initiation complex required for ribosome binding to mRNA and promote translation. Translation of ferritin IRE mRNAs is regulated by iron through iron responsive elements (IREs) and iron regulatory protein (IRP). The noncoding IRE stem-loop (30-nt) structure control synthesis of proteins in iron trafficking, cell cycling, and nervous system function. High cellular iron concentrations promote IRE RNA binding to ribosome and initiation factors, and allow synthesis of ferritin. In vitro translation assay was performed in depleted wheat germ lysate with supplementation of initiation factors. Fluorescence spectroscopy was used to characterize eIF4F/IRE binding. Eukaryotic initiation factor eIF4G increases the translation of ferritin through binding to stem loop structure of iron responsive elements mRNA in the 5'-untranslated region. Our translation experiment demonstrated that exogenous addition of eIF4G selectively enhanced the translation of ferritin IRE RNA in depleted WG lysate. However, eIF4G facilitates capped IRE RNA translation significantly higher than uncapped IRE RNA translation. Addition of iron with eIF4G to depleted WG lysate significantly enhanced translation for both IRE mRNA (capped and uncapped), confirming the contribution of eIF4G and iron as a potent enhancer of ferritin IRE mRNA translation. Fluorescence data revealed that ferritin IRE strongly interacts to eIF4G (Kd = 63 nM), but not eIF4E. Further equilibrium studies showed that iron enhanced (~4-fold) the ferritin IRE binding to eIF4G. The equilibrium binding effects of iron on ferritin IRE RNA/eIFs interaction and the temperature dependence of this reaction were measured and compared. The Kd values for the IRE binding to eIF4G ranging from 18.2 nM to 63.0 nM as temperature elevated from 5 °C to 25 °C, while the presence of iron showed much stronger affinity over the same range of temperatures. Thermodynamic parameter revealed that IRE RNA binds to eIF4G with ΔH = -42.6 ± 3.3 kJ. mole-1, ΔS = -11.5 ± 0.4 J. mole-1K-1, and ΔG = -39.2 ± 2.7 kJ. mole-1, respectively. Furthermore, addition of iron significantly changed the values of thermodynamic parameters, favoring stable complex formation, thus favoring efficient protein synthesis. This study first time demonstrate the participation of eIF4G in ferritin IRE mRNA translation. eIF4G specifically interacts with ferritin IRE RNA and promotes eIF4G-dependent translation.

Nuclear speckle integrity and function require TAO2 kinase

Nuclear speckles are non-membrane-bound organelles known as storage sites for messenger RNA (mRNA) processing and splicing factors. More recently, nuclear speckles have also been implicated in splicing and export of a subset of mRNAs, including the influenza virus M mRNA that encodes proteins required for viral entry, trafficking, and budding. However, little is known about how nuclear speckles are assembled or regulated. Here, we uncovered a role for the cellular protein kinase TAO2 as a constituent of nuclear speckles and as a factor required for the integrity of these nuclear bodies and for their functions in pre-mRNA splicing and trafficking. We found that a nuclear pool of TAO2 is localized at nuclear speckles and interacts with nuclear speckle factors involved in RNA splicing and nuclear export, including SRSF1 and Aly/Ref. Depletion of TAO2 or inhibition of its kinase activity disrupts nuclear speckle structure, decreasing the levels of several proteins involved in nuclear speckle assembly and splicing, including SC35 and SON. Consequently, splicing and nuclear export of influenza virus M mRNA were severely compromised and caused a disruption in the virus life cycle. In fact, low levels of TAO2 led to a decrease in viral protein levels and inhibited viral replication. Additionally, depletion or inhibition of TAO2 resulted in abnormal expression of a subset of mRNAs with key roles in viral replication and immunity. Together, these findings uncovered a function of TAO2 in nuclear speckle formation and function and revealed host requirements and vulnerabilities for influenza infection.

Untangling the Conformational Plasticity of V66M Human proBDNF Polymorphism as a Modifier of Psychiatric Disorder Susceptibility.

The human genetic variant BDNF (V66M) represents the first example of neurotrophin family member that has been linked to psychiatric disorders. In order to elucidate structural differences that account for the effects in cognitive function, this hproBDNF polymorph was expressed, refolded, purified, and compared directly to the WT variant for the first time for differences in their 3D structures by DSF, limited proteolysis, FT-IR, and SAXS measurements in solution. Our complementary studies revealed a deep impact of V66M polymorphism on hproBDNF conformations in solution. Although the mean conformation in solution appears to be more compact in the V66M variant, overall, we demonstrated a large increase in flexibility in solution upon V66M mutation. Thus, considering that plasticity in IDR is crucial for protein function, the observed alterations may be related to the functional alterations in hproBDNF binding to its receptors p75NTR, sortilin, HAP1, and SorCS2. These effects can provoke altered intracellular neuronal trafficking and/or affect proBDNF physiological functions, leading to many brain-associated diseases and conditions such as cognitive impairment and anxiety. The structural alterations highlighted in the present study may pave the way to the development of drug discovery strategies to provide greater therapeutic responses and of novel pharmacologic strategy in human populations with this common polymorphism, ultimately guiding personalized medicine for neuropsychiatric disorders.

Membrane trafficking functions of the ANTH/ENTH/VHS domain-containing proteins in plants.

Subcellular localization of proteins acting on the endomembrane system is primarily regulated via membrane trafficking. To obtain and maintain the correct protein composition of the plasma membrane and membrane-bound organelles, the loading of selected cargos into transport vesicles is critically regulated at donor compartments by adaptor proteins binding to the donor membrane, the cargo molecules and the coat-protein complexes, including the clathrin coat. The ANTH/ENTH/VHS domain-containing protein superfamily generally comprises a structurally related ENTH, ANTH, or VHS domain in the N-terminal region and a variable C-terminal region, which is thought to act as an adaptor during transport vesicle formation. This protein family is involved in various plant processes, including pollen tube growth, abiotic stress response and development. In this review, we provide an overview of the recent findings on ANTH/ENTH/VHS domain-containing proteins in plants.

LFA1 Activation: Insights from a Single-Molecule Approach.

Integrin LFA1 is a cell adhesion receptor expressed exclusively in leukocytes, and plays crucial roles in lymphocyte trafficking, antigen recognition, and effector functions. Since the discovery that the adhesiveness of LFA1 can be dynamically changed upon stimulation, one challenge has been understanding how integrins are regulated by inside-out signaling coupled with macromolecular conformational changes, as well as ligand bindings that transduce signals from the extracellular domain to the cytoplasm in outside-in signaling. The small GTPase Rap1 and integrin adaptor proteins talin1 and kindlin-3 have been recognized as critical molecules for integrin activation. However, their cooperative regulation of integrin adhesiveness in lymphocytes requires further research. Recent advances in single-molecule imaging techniques have revealed dynamic molecular processes in real-time and provided insight into integrin activation in cellular environments. This review summarizes integrin regulation and discusses new findings regarding the bidirectionality of LFA1 activation and signaling processes in lymphocytes.

Mucosal-Associated Invariant T (MAIT) cells are highly activated in duodenal tissue of humans with Vibrio cholerae O1 infection: A preliminary report.

Mucosal-associated invariant T (MAIT) cells are unconventional T lymphocytes with a semi-conserved TCRα, activated by the presentation of vitamin B metabolites by the MHC-I related protein, MR1, and with diverse innate and adaptive effector functions. The role of MAIT cells in acute intestinal infections, especially at the mucosal level, is not well known. Here, we analyzed the presence and phenotype of MAIT cells in duodenal biopsies and paired peripheral blood samples, in patients during and after culture-confirmed Vibrio cholerae O1 infection. Immunohistochemical staining of duodenal biopsies from cholera patients (n = 5, median age 32 years, range 26-44, 1 female) identified MAIT cells in the lamina propria of the crypts, but not the villi. By flow cytometry (n = 10, median age 31 years, range 23-36, 1 female), we showed that duodenal MAIT cells are more activated than peripheral MAIT cells (p < 0.01 across time points), although there were no significant differences between duodenal MAIT cells at day 2 and day 30. We found fecal markers of intestinal permeability and inflammation to be correlated with the loss of duodenal (but not peripheral) MAIT cells, and single-cell sequencing revealed differing T cell receptor usage between the duodenal and peripheral blood MAIT cells. In this preliminary report limited by a small sample size, we show that MAIT cells are present in the lamina propria of the duodenum during V. cholerae infection, and more activated than those in the blood. Future work into the trafficking and tissue-resident function of MAIT cells is warranted.

A phosphorylation network controls the stability of alpha‐arrestins Aly1/Art6 and Aly2/Art3

Selective protein trafficking controls the repertoire of membrane proteins at the cell surface. This contingent of surface proteins regulates nutrient/metabolite balance and response to extracellular cues. Environmental changes trigger cellular signaling that drives transitions in the plasma membrane proteome. These alterations are achieved in no small part through regulation of the α‐arrestins, an emergent and powerful class of protein trafficking adaptors. The α‐arrestins are largely cytosolic proteins that are transiently recruited to membranes via binding to membrane protein motifs. α‐Arrestins bring with them a ubiquitin ligase, which stimulates ubiquitination and subsequent endocytosis of membrane proteins. Phosphorylation of α‐arrestins is key to regulating their trafficking function; dephosphorylated α‐arrestins are generally ‘active’ endocytic adaptors. While some kinases and phosphatases for α‐arrestins are known, the degree of α‐arrestins phosphorylation is staggering, with &gt;40 phosphorylation sites identified for a single α‐arrestin, and suggests we still have much to learn about α‐arrestins regulation. We sought to determine what kinases and phosphates regulate paralogous α‐arrestins Aly1 and Aly2 using a genetic screen in yeast. Using an array of all known non‐essential kinase and phosphatase deletions, we determined which of these influenced α‐arrestin‐mediated resistance to rapamycin, an inhibitor of the TORC1 nutrient‐sensing kinase. We identified a large cohort of kinases and phosphates that influenced Aly‐dependent phenotypes, causing electrophoretic mobility changes and, in many cases, diminishing the abundance of these α‐arrestins. We focused our studies on the Sit4 protein phosphatase, a key regulator downstream of TORC1 signaling able to influence other α‐arrestins, identified in our screen. Strikingly, Aly1 and Aly2 were hyperphosphorylated and destabilized in the absence of Sit4, suggesting that excessive phosphorylation may promote degradation of these α‐arrestins. For Aly2, but not Aly1, degradation in the sit4∆ cells could be reversed by loss of the vacuolar protease Pep4, indicating that vacuolar degradation predominates for Aly2 under these conditions. Loss of the Npr1 kinase in sit4∆ cells restored Aly protein abundances and reversed the hyperphosphorylation, demonstrating that this kinase is responsible for the excess Aly phosphorylation in sit4∆ cells. This is a remarkable finding as typically Npr1 is considered inactive in sit4∆, however, we suggest that Npr1 is selectively active in the absence of Sit4, able to modify some substrates but not others. We define new features of TORC1 signaling in regulating α‐arrestin phosphorylation and stability.

Interactions of the intrinsically disordered selenoprotein S with a small GTPase regulator

Selenoproteins are a family of enzymes that employ the rare amino acid selenocysteine to catalyze chemical reactions. Among them, selenoprotein S stands out because the selenocysteine is positioned in an intrinsically disordered segment. The physiological function of this enzyme is unknown, although it was shown to take part in ER homeostasis by mediating protein degradation and may also have a role in vesicle trafficking, lipid metabolism, and management of oxidative stress. To elucidate its function in vesicle trafficking, we demonstrate that selenoprotein S binds the nucleotide exchange regulator of small GTPases, SmgGDS, a regulator of the Ras and Rho family members. Curiously, both selenoprotein S and SmgGDS are hijacked by non‐structural proteins of SARS‐CoV‐2, along with other proteins involved in maintaining ER homeostasis and regulation of the secretory pathway. To investigate this link, we have characterized the interactions between selenoprotein S and SmgGDS. We show that the interaction requires the hydrophobic segment of selenoprotein S, previously thought to be transmembrane. We describe biochemical assays to examine the putative role of selenoprotein S in modulating SmgGDS function and whether it accelerates the rate release of GTPases from SmgGDS.