Bacterial Metabolism and Transport Genes Are Associated with the Preference of Drosophila melanogaster for Dietary Yeast.

Many animal traits are influenced by their associated microorganisms ("microbiota"). To expand our understanding of the relationship between microbial genotype and host phenotype, we report an analysis of the influence of the microbiota on the dietary preference of the fruit fly Drosophila melanogaster. First, we confirmed through experiments on flies reared bacteria-free ("axenic") or in monoassociation with two different strains of bacteria that the microbiota significantly influences fruit fly dietary preference across a range of ratios of dietary yeast:dietary glucose.

Opioid Use Disorder Curriculum: Preclerkship Pharmacology Case-Based Learning Session.

Introduction: During the first year of the COVID-19 pandemic, over 93,000 Americans lost their lives to a preventable overdose. Medications for opioid use disorder (OUD) have been shown to decrease mortality in OUD but are underutilized. Through this case-based learning exercise, first-year medical students applied physiologic and pharmacologic principles to the diagnosis and treatment of OUD.

Atg27p localization is clathrin- and Ent3p/5p-dependent.

The autophagy-related protein Atg27p has been previously shown to localize to the autophagy-specific pre-autophagosomal structure (PAS) as well as to several organelles, including the late Golgi, the vacuolar membrane, and the endosome. Given that Atg27p localization to the vacuolar membrane in particular has been shown to be dependent on both its C-terminal tyrosine sorting motif and the AP-3 adaptor, and that Atg27p can be found in clathrin-coated vesicles, we set out to determine whether Atg27p localization inside cells is dependent on clathrin or on any of its cargo adaptors. We report that Atg27p localization is clathrin- and Ent3p/5p-dependent.

Atg27p co-fractionates with clathrin-coated vesicles in budding yeast.

Atg27p, a single-pass transmembrane protein that functions in autophagy, localizes to a variety of cellular compartments including the pre-autophagosomal structure, late Golgi, vacuolar membrane, as well as early and late endosomes. Its cytoplasmic C-terminus contains a tyrosine sorting motif that allows for its transport to the vacuolar membrane and an additional sequence that allows for its retrieval from the vacuolar membrane to the endosome. Since clathrin is well known to mediate vesicular transport in the endomembrane system, the trafficking of Atg27p and its tyrosine sorting motif suggested that it might be trafficked inside clathrin-coated vesicles (CCVs).

Microbiome composition shapes rapid genomic adaptation of .

Population genomic data has revealed patterns of genetic variation associated with adaptation in many taxa. Yet understanding the adaptive process that drives such patterns is challenging; it requires disentangling the ecological agents of selection, determining the relevant timescales over which evolution occurs, and elucidating the genetic architecture of adaptation. Doing so for the adaptation of hosts to their microbiome is of particular interest with growing recognition of the importance and complexity of host-microbe interactions.

Identification of Suppressor of Clathrin Deficiency-1 () and Its Connection to Clathrin-Mediated Endocytosis in .

Clathrin is a major coat protein involved in vesicle formation during endocytosis and transport in the endosomal/trans Golgi system. Clathrin is required for normal growth of yeast and in some genetic backgrounds deletion of the clathrin heavy chain gene () is lethal. Our lab defined a locus referred to as " uppressor of lathrin eficiency" ().

Creating a chimeric clathrin heavy chain that functions independently of yeast clathrin light chain.

Clathrin facilitates vesicle formation during endocytosis and sorting in the trans-Golgi network (TGN)/endosomal system. Unlike in mammals, yeast clathrin function requires both the clathrin heavy (CHC) and clathrin light (CLC) chain, since Chc1 does not form stable trimers without Clc1. To further delineate clathrin subunit functions, we constructed a chimeric CHC protein (Chc-YR) , which fused the N-terminus of yeast CHC (1-1312) to the rat CHC residues 1318-1675, including the CHC trimerization region.

Auxilin facilitates membrane traffic in the early secretory pathway.

Coat protein complexes contain an inner shell that sorts cargo and an outer shell that helps deform the membrane to give the vesicle its shape. There are three major types of coated vesicles in the cell: COPII, COPI, and clathrin. The COPII coat complex facilitates vesicle budding from the endoplasmic reticulum (ER), while the COPI coat complex performs an analogous function in the Golgi.

Atg27 tyrosine sorting motif is important for its trafficking and Atg9 localization.

During autophagy, the transmembrane protein Atg27 facilitates transport of the major autophagy membrane protein Atg9 to the preautophagosomal structure (PAS). To better understand the function of Atg27 and its relationship with Atg9, Atg27 trafficking and localization were examined. Atg27 localized to endosomes and the vacuolar membrane, in addition to previously described PAS, Golgi and Atg9-positive structures.

Bem3, a Cdc42 GTPase-activating protein, traffics to an intracellular compartment and recruits the secretory Rab GTPase Sec4 to endomembranes.

Cell polarity is essential for many cellular functions including division and cell-fate determination. Although RhoGTPase signaling and vesicle trafficking are both required for the establishment of cell polarity, the mechanisms by which they are coordinated are unclear. Here, we demonstrate that the yeast RhoGAP (GTPase activating protein), Bem3, is targeted to sites of polarized growth by the endocytic and recycling pathways.

Novel interaction between the co-chaperone Cdc37 and Rho GTPase exchange factor Vav3 promotes androgen receptor activity and prostate cancer growth.

Elevated androgen receptor (AR) activity in castration-resistant prostate cancer may occur through increased levels of AR co-activator proteins. Vav3, a guanine nucleotide exchange factor, is up-regulated following progression to castration resistance in preclinical models and is overexpressed in a significant number of human prostate cancers. Vav3 is a novel co-activator of the AR.

Role of Scd5, a protein phosphatase-1 targeting protein, in phosphoregulation of Sla1 during endocytosis.

Phosphorylation regulates assembly and disassembly of proteins during endocytosis. In yeast, Prk1 and Ark1 phosphorylate factors after vesicle internalization leading to coat disassembly. Scd5, a protein phosphatase-1 (PP1)-targeting subunit, is proposed to regulate dephosphorylation of Prk1/Ark1 substrates to promote new rounds of endocytosis.

Getting in touch with the clathrin terminal domain.

The N-terminal domain (TD) of the clathrin heavy chain is folded into a seven-bladed β-propeller that projects inward from the polyhedral outer clathrin coat. As the most membrane-proximal portion of assembled clathrin, the TD is a major protein-protein interaction node. Contact with the TD β-propeller occurs through short peptide sequences typically located within intrinsically disordered segments of coat components that usually are elements of the membrane-apposed, inner 'adaptor' coat layer.

Lessons from yeast for clathrin-mediated endocytosis.

Clathrin-mediated endocytosis (CME) is the major pathway for internalization of membrane proteins from the cell surface. Half a century of studies have uncovered tremendous insights into how a clathrin-coated vesicle is formed. More recently, the advent of live-cell imaging has provided a dynamic view of this process.

Clathrin light chain directs endocytosis by influencing the binding of the yeast Hip1R homologue, Sla2, to F-actin.

The role of clathrin light chain (CLC) in clathrin-mediated endocytosis is not completely understood. Previous studies showed that the CLC N-terminus (CLC-NT) binds the Hip1/Hip1R/Sla2 family of membrane/actin-binding factors and that overexpression of the CLC-NT in yeast suppresses endocytic defects of clathrin heavy-chain mutants. To elucidate the mechanistic basis for this suppression, we performed synthetic genetic array analysis with a clathrin CLC-NT deletion mutation (clc1-Δ19-76).

Calmodulin dissociation regulates Myo5 recruitment and function at endocytic sites.

Myosins-I are conserved proteins that bear an N-terminal motor head followed by a Tail Homology 1 (TH1) lipid-binding domain. Some myosins-I have an additional C-terminal extension (C(ext)) that promotes Arp2/3 complex-dependent actin polymerization. The head and the tail are separated by a neck that binds calmodulin or calmodulin-related light chains.

The F-BAR protein Syp1 negatively regulates WASp-Arp2/3 complex activity during endocytic patch formation.

Background: Actin polymerization by Arp2/3 complex must be tightly regulated to promote clathrin-mediated endocytosis. Although many Arp2/3 complex activators have been identified, mechanisms for its negative regulation have remained more elusive. To address this, we analyzed the yeast arp2-7 allele, which is biochemically unique in causing unregulated actin assembly in vitro in the absence of Arp2/3 activators.

Clathrin functions in the absence of the terminal domain binding site for adaptor-associated clathrin-box motifs.

Clathrin is involved in vesicle formation in the trans-Golgi network (TGN)/endosomal system and during endocytosis. Clathrin recruitment to membranes is mediated by the clathrin heavy chain (HC) N-terminal domain (TD), which forms a seven-bladed beta-propeller. TD binds membrane-associated adaptors, which have short peptide motifs, either the clathrin-box (CBM) and/or the W-box; however, the importance of the TD binding sites for these motifs has not been tested in vivo.

Novel function of clathrin light chain in promoting endocytic vesicle formation.

Clathrin-mediated endocytosis is a major pathway for uptake of lipid and protein cargo at the plasma membrane. The lattices of clathrin-coated pits and vesicles are comprised of triskelions, each consisting of three oligomerized heavy chains (HC) bound by a light chain (LC). In addition to binding HC, LC interacts with members of the Hip1/R family of endocytic proteins, including the budding yeast homologue, Sla2p.

Clathrin is important for normal actin dynamics and progression of Sla2p-containing patches during endocytosis in yeast.

Clathrin is a major vesicle coat protein involved in receptor-mediated endocytosis. In yeast and higher eukaryotes, clathrin is recruited to the plasma membrane during the early stage of endocytosis along with clathrin-associated adaptors. As coated pits undergo maturation, a burst of actin polymerization accompanies and helps drive vesicle internalization.

Cortical recruitment and nuclear-cytoplasmic shuttling of Scd5p, a protein phosphatase-1-targeting protein involved in actin organization and endocytosis.

Scd5p regulates endocytosis and cortical actin organization as a targeting subunit for the Ser/Thr protein phosphatase-1 (PP1) in yeast. To identify localization signals in Scd5p required for cell surface recruitment, visualization of GFP-tagged Scd5 truncations and deletions was performed. Scd5p contains a PP1 binding site, a 3-repeat region of 20 amino acids (3R), and a 9-repeat region of 12 amino acids (9R).

RanBPM is an L1-interacting protein that regulates L1-mediated mitogen-activated protein kinase activation.

A yeast two-hybrid screen using the last 28 amino acids of the cytoplasmic domain of the neural cell adhesion molecule L1 identified RanBPM as an L1-interacting protein. RanBPM associates with L1 in vivo and the N-terminal region of RanBPM (N-RanBPM), containing the SPRY domain, is sufficient for the interaction with L1 in a glutathione S-transferase pull-down assay. L1 antibody patching dramatically changes the subcellular localization of N-RanBPM in transfected COS cells.

In vivo dynamics of clathrin and its adaptor-dependent recruitment to the actin-based endocytic machinery in yeast.

Clathrin-mediated transport is a major pathway for endocytosis. However, in yeast, where cortical actin patches are essential for endocytosis, plasma membrane-associated clathrin has never been observed. Using live cell imaging, we demonstrate cortical clathrin in association with the actin-based endocytic machinery in yeast.

The actin-regulating kinase Prk1p negatively regulates Scd5p, a suppressor of clathrin deficiency, in actin organization and endocytosis.

Endocytosis is a dynamic process requiring a network of interacting proteins that assemble and disassemble during cargo capture and vesicle formation. A major mechanism for regulation of this process involves the reversible phosphorylation of endocytic factors. Recently, members of a new kinase family, the Ark/Prk kinases, which include mammalian AAK1 and GAK as well as yeast Prk1p, Ark1p, and Akl1p, were shown to regulate components of the endocytic machinery.