Ectopic expression of MHC class II genes (RT1.B(I) beta/alpha) in rat hepatocytes in vivo and in culture can be elicited by treatment with the pregnane X receptor agonists pregnenolone 16 alpha-carbonitrile and dexamethasone

The synthetic steroid, pregnenolone-16-alpha-carbonitrile (PCN), has served for decades as a probe for a postulated series of hepatic defenses activated under situations of environmental "stress". PCN, an antiglucocorticoid, and also such glucocorticoids as dexamethasone (Dex) appear to stimulate hepatic metabolism and elimination of xenobiotics by binding to the nuclear pregnane X receptor (PXR) which then interacts with a distinct DNA response element associated with induction of cytochrome P450 3A genes. To explore the full domain of genes controlled by PCN/PXR, we used differential display to detect rat liver mRNA species selectively induced by PCN or by Dex. Sequence analysis identified one of many PCN induced cDNA fragments as RT1.B(I)beta, a member of the major histocompatability class II (MHC) gene family usually found only in antigen presenting cells. Northern blot analysis of RNA from rat liver or from cultured hepatocytes confirmed that amounts of RT1.B(I)beta mRNA and also of its companion gene, RT1.B(I)alpha mRNA, became readily detectable within 3-6 hours following treatment with PCN or Dex, whereas no induction was observed in spleen RNA. Induction by PCN of RT1.B(I)beta immunoreactive protein was localized to the hepatocytes as judged by immunofluorescence. We conclude that ectopic expression of MHC II genes, an unprecedented effect of steroids or drugs, is rapidly evoked by PCN acting on the liver, directly. The concept of a set of genes coordinately controlled to maintain homeostasis in parenchymal tissues during toxic stress must now be extended to include the immune system.     

Regulators of yeast endocytosis identified by systematic quantitative analysis

Endocytosis of receptors at the plasma membrane is controlled by a complex mechanism that includes clathrin, adaptors, and actin regulators. Many of these proteins are conserved in yeast yet lack observable mutant phenotypes, which suggests that yeast endocytosis may be subject to different regulatory mechanisms. Here, we have systematically defined genes required for internalization using a quantitative genome-wide screen that monitors localization of the yeast vesicle-associated membrane protein (VAMP)/synaptobrevin homologue Snc1. Genetic interaction mapping was used to place these genes into functional modules containing known and novel endocytic regulators, and cargo selectivity was evaluated by an array-based comparative analysis. We demonstrate that clathrin and the yeast AP180 clathrin adaptor proteins have a cargo-specific role in Snc1 internalization. We additionally identify low dye binding 17 (LDB17) as a novel conserved component of the endocytic machinery. Ldb17 is recruited to cortical actin patches before actin polymerization and regulates normal coat dynamics and actin assembly. Our findings highlight the conserved machinery and reveal novel mechanisms that underlie endocytic internalization.     

New regulators of a high affinity Ca2+ influx system revealed through a genome-wide screen in yeast

The bakers' yeast Saccharomyces cerevisiae utilizes a high affinity Ca(2+) influx system (HACS) to survive assaults by mating pheromones, tunicamycin, and azole-class antifungal agents. HACS consists of two known subunits, Cch1 and Mid1, that are homologous and analogous to the catalytic α-subunits and regulatory α2δ-subunits of mammalian voltage-gated calcium channels, respectively. To search for additional subunits and regulators of HACS, a collection of gene knock-out mutants was screened for abnormal uptake of Ca(2+) after exposure to mating pheromone or to tunicamycin. The screen revealed that Ecm7 is required for HACS function in most conditions. Cycloheximide chase experiments showed that Ecm7 was stabilized by Mid1, and Mid1 was stabilized by Cch1 in non-signaling conditions, suggesting they all interact. Ecm7 is a member of the PMP-22/EMP/MP20/Claudin superfamily of transmembrane proteins that includes γ-subunits of voltage-gated calcium channels. Eleven additional members of this superfamily were identified in yeast, but none was required for HACS activity in response to the stimuli. Remarkably, many dozens of genes involved in vesicle-mediated trafficking and protein secretion were required to prevent spontaneous activation of HACS. Taken together, the findings suggest that HACS and calcineurin monitor performance of the membrane trafficking system in yeasts and coordinate compensatory processes. Conservation of this quality control system in Candida glabrata suggests that many pathogenic species of fungi may utilize HACS and calcineurin to resist azoles and other compounds that target membrane biosynthesis.     

The Function of Yeast Epsin and Ede1 Ubiquitin-Binding Domains During Receptor Internalization

The formation of a primary endocytic vesicle is a dynamic process involving the transient organization of adaptor and scaffold proteins at the plasma membrane. Epsins and Eps15-like proteins are ubiquitin-binding proteins that act early in this process. The yeast epsins, Ent1 and Ent2, carry functional ubiquitin-interacting motifs (UIMs), whereas the yeast Eps15-like protein, Ede1, has a C-terminal ubiquitin-associated (UBA) domain. Analysis of mutants lacking early endocytic adaptors reveals that the ubiquitin-binding domains (UBDs) of Ent2 and Ede1 are likely to function primarily to mediate protein–protein interactions between components of the early endocytic machinery. Cells that lack epsin and Ede1 UBDs are able to internalize activated, ubiquitinated receptors. Furthermore, under conditions in which epsin UIMs are important for receptor internalization, receptors internalized via both ubiquitin-dependent and ubiquitin-independent signals require the UIMs, indicating that UIM function is not restricted to ubiquitinated receptors. Epsin UIMs share function with non-UBD protein–protein interaction motifs in Ent2 and Ede1, and the Ede1 UBA domain appears to negatively regulate interactions between endocytic proteins. Together, our results suggest that the ubiquitin-binding domains within the yeast epsin Ent2 and Ede1 are involved in the formation and regulation of the endocytic network.     

Existence of a novel clathrin-independent endocytic pathway in yeast that depends on Rho1 and formin

Yeast is a powerful model organism for dissecting the temporal stages and choreography of the complex protein machinery during endocytosis. The only known mechanism for endocytosis in yeast is clathrin-mediated endocytosis, even though clathrin-independent endocytic pathways have been described in other eukaryotes. Here, we provide evidence for a clathrin-independent endocytic pathway in yeast. In cells lacking the clathrin-binding adaptor proteins Ent1, Ent2, Yap1801, and Yap1802, we identify a second endocytic pathway that depends on the GTPase Rho1, the downstream formin Bni1, and the Bni1 cofactors Bud6 and Spa2. This second pathway does not require components of the better-studied endocytic pathway, including clathrin and Arp2/3 complex activators. Thus, our results reveal the existence of a second pathway for endocytosis in yeast, which suggests similarities with the RhoA-dependent endocytic pathways of mammalian cells.     

Interaction of the endocytic scaffold protein Pan1 with the type I myosins contributes to the late stages of endocytosis

The yeast endocytic scaffold Pan1 contains an uncharacterized proline-rich domain (PRD) at its carboxy (C)-terminus. We report that the pan1-20 temperature-sensitive allele has a disrupted PRD due to a frame-shift mutation in the open reading frame of the domain. To reveal redundantly masked functions of the PRD, synthetic genetic array screens with a pan1DeltaPRD strain found genetic interactions with alleles of ACT1, LAS17 and a deletion of SLA1. Through a yeast two-hybrid screen, the Src homology 3 domains of the type I myosins, Myo3 and Myo5, were identified as binding partners for the C-terminus of Pan1. In vitro and in vivo assays validated this interaction. The relative timing of recruitment of Pan1-green fluorescent protein (GFP) and Myo3/5-red fluorescent protein (RFP) at nascent endocytic sites was revealed by two-color real-time fluorescence microscopy; the type I myosins join Pan1 at cortical patches at a late stage of internalization, preceding the inward movement of Pan1 and its disassembly. In cells lacking the Pan1 PRD, we observed an increased lifetime of Myo5-GFP at the cortex. Finally, Pan1 PRD enhanced the actin polymerization activity of Myo5-Vrp1 complexes in vitro. We propose that Pan1 and the type I myosins interactions promote an actin activity important at a late stage in endocytic internalization.     

Endocytic adaptors: recruiters, coordinators and regulators

Clathrin-dependent endocytosis allows cells to bring plasma membrane and extracellular molecules into the cell. Forming a clathrin-coated vesicle requires the sequential action of numerous factors, beginning with endocytic adaptors. Adaptors are thought to initiate the process in two ways: by selecting cargo for packaging into the vesicle and assembling the clathrin coat and other components necessary to shape the vesicle. Here, we review recent work focusing on the sequential and cooperative interactions of adaptors with their binding partners, and how adaptors contribute to initial stages of endocytic internalization. The regulation of adaptors might be a key step for controlling endocytosis, and thus aid in homeostasis and cell physiology.     

Interaction between Epsin/Yap180 adaptors and the scaffolds Ede1/Pan1 is required for endocytosis

The spatial and temporal regulation of the interactions among the approximately 60 proteins required for endocytosis is under active investigation in many laboratories. We have identified the interaction between monomeric clathrin adaptors and endocytic scaffold proteins as a critical prerequisite for the recruitment and/or spatiotemporal dynamics of endocytic proteins at early and late stages of internalization. Quadruple deletion yeast cells (DeltaDeltaDeltaDelta) lacking four putative adaptors, Ent1/2 and Yap1801/2 (homologues of epsin and AP180/CALM proteins), with a plasmid encoding Ent1 or Yap1802 mutants, have defects in endocytosis and growth at 37 degrees C. Live-cell imaging revealed that the dynamics of the early- and late-acting scaffold proteins Ede1 and Pan1, respectively, depend upon adaptor interactions mediated by adaptor asparagine-proline-phenylalanine motifs binding to scaffold Eps15 homology domains. These results suggest that adaptor/scaffold interactions regulate transitions from early to late events and that clathrin adaptor/scaffold protein interaction is essential for clathrin-mediated endocytosis.     

A Highlights from MBoC Selection: The alternate AP-1 adaptor subunit Apm2 interacts with the Mil1 regulatory protein and confers differential cargo sorting

Heterotetrameric adaptor protein complexes are important mediators of cargo protein sorting in clathrin-coated vesicles. The cell type–specific expression of alternate μ chains creates distinct forms of AP-1 with altered cargo sorting, but how these subunits confer differential function is unclear. Whereas some studies suggest the μ subunits specify localization to different cellular compartments, others find that the two forms of AP-1 are present in the same vesicle but recognize different cargo. Yeast have two forms of AP-1, which differ only in the μ chain. Here we show that the variant μ chain Apm2 confers distinct cargo-sorting functions. Loss of Apm2, but not of Apm1, increases cell surface levels of the v-SNARE Snc1. However, Apm2 is unable to replace Apm1 in sorting Chs3, which requires a dileucine motif recognized by the γ/σ subunits common to both complexes. Apm2 and Apm1 colocalize at Golgi/early endosomes, suggesting that they do not associate with distinct compartments. We identified a novel, conserved regulatory protein that is required for Apm2-dependent sorting events. Mil1 is a predicted lipase that binds Apm2 but not Apm1 and contributes to its membrane recruitment. Interactions with specific regulatory factors may provide a general mechanism to diversify the functional repertoire of clathrin adaptor complexes.     

Clathrin-independent endocytosis: A cargo-centric view

Clathrin-independent endocytosis occurs in all cells and interest in this mode of cellular entry has grown. Although this form of endocytosis was first described for entry of bacterial toxins, here we focus our attention on the endogenous cell surface “cargo” proteins that enter cells by this mechanism. The cargo proteins entering by this mechanism are varied and include nutrient transporters, ion channels, cell adhesion molecules and proteins associated with the immune system. Despite the apparent lack of selection at the cell surface, we provide some examples of specific sorting of these cargo proteins after entry, leading to distinct itineraries and cellular fates.