Differential selection of Golgi proteins by COPII Sec24 isoforms in procyclic Trypanosoma brucei

The Sec24 subunit of the coat protein complex II (COPII) has been implicated in selecting newly synthesized cargo from the endoplasmic reticulum (ER) for delivery to the Golgi. The protozoan parasite, Trypanosoma brucei, contains two paralogs, TbSec24.1 and TbSec24.2, which were depleted using RNA interference in the insect form of the parasite. Depletion of either TbSec24.1 or TbSec24.2 resulted in growth arrest and modest inhibition of anterograde transport of the putative Golgi enzyme, TbGntB, and the secretory marker, BiPNAVRG-HA9. In contrast, depletion of TbSec24.1, but not TbSec24.2, led to reversible mislocalization of the Golgi stack proteins, TbGRASP and TbGolgin63. The latter accumulated in the ER. The localization of the COPI coatomer subunit, TbεCOP, and the trans Golgi network (TGN) protein, TbGRIP70, was largely unaffected, although the latter was preferentially lost from those Golgi that were not associated with the bilobe, a structure previously implicated in Golgi biogenesis. Together, these data suggest that TbSec24 paralogs can differentiate among proteins destined for the Golgi.     

Genetic Analysis of Yeast Sec24p Mutants Suggests Cargo Binding Is Not Co‐operative during ER Export

Many eukaryotic secretory proteins are selected for export from the endoplasmic reticulum (ER) through their interaction with the Sec24p subunit of the coat protein II (COPII) coat. Three distinct cargo‐binding sites on yeast Sec24p have been described by biochemical, genetic and structural studies. Each site recognizes a limited set of peptide motifs or a folded structural domain, however, the breadth of cargo recognized by a given site and the dynamics of cargo engagement remain poorly understood. We aimed to gain further insight into the broader molecular function of one of these cargo‐binding sites using a non‐biased genetic approach. We exploited the in vivo lethality associated with mutation of the Sec24p B‐site to identify genes that suppress this phenotype when overexpressed. We identified SMY2 as a general suppressor that rescued multiple defects in Sec24p, and SEC22 as a specific suppressor of two adjacent cargo‐binding sites, raising the possibility of allosteric regulation of these domains. We generated a novel set of mutations in Sec24p that distinguish these two sites and examined the ability of Sec22p to rescue these mutations. Our findings suggest that co‐operativity does not influence cargo capture at these sites, and that Sec22p rescue occurs via its function as a retrograde SNARE.     

Interaction of the K(+)-channel KAT1 with the coat protein complex II coat component Sec24 depends on a di-acidic endoplasmic reticulum export motif

The correct functioning of ion channels depends not only on the control of their activity but also on the regulation of the number of channels in the membrane. For example, it has been proposed that the density of the plant K(+)-channel KAT1 may be adjusted by controlling its export from its site of synthesis, the endoplasmic reticulum (ER). Efficient transport of the channel to the plasma membrane was found to depend on a di-acidic ER export signal in the C-terminus of the protein. Studies in yeast and mammals indicate that di-acidic ER export motifs are essential for enrichment of proteins into ER-derived coat protein complex II (COPII) vesicles and are recognized by Sec24 a component of the COPII coat. To investigate whether similar mechanisms also exist in plants we have analysed the interaction of KAT1 with Sec24 in vivo using fluorescence resonance energy transfer (FRET) measurements in Vicia faba guard cells. These measurements revealed a FRET signal between KAT1 and Sec24 fused to the cyan fluorescent protein and the yellow fluorescent protein, respectively, indicating an interaction between KAT1 and Sec24. The FRET signal only occurred in the perinuclear region of the ER and was dependent on the di-acidic ER export motif of KAT1. Together, the results point to a highly conserved mechanism for ER export of KAT1 whereby the channel is recruited into COPII vesicles via binding of the di-acidic motif to Sec24.     

Endoplasmic reticulum exit of a secretory glycoprotein in the absence of sec24p family proteins in yeast

Glycoproteins exit the endoplasmic reticulum (ER) of the yeast Saccharomyces cerevisiae in coat protein complex II (COPII) coated vesicles. The coat consists of the essential proteins Sec23p, Sec24p, Sec13p, Sec31p, Sar1p and Sec16p. Sec24p and its two nonessential homologues Sfb2p and Sfb3p have been suggested to serve in cargo selection. Using temperature-sensitive sec24-1 mutants, we showed previously that a secretory glycoprotein, Hsp150, does not require functional Sec24p for ER exit. Deletion of SFB2, SFB3 or both from wild type or the deletion of SFB2 from sec24-1 cells did not affect Hsp150 transport. SFB3 deletion has been reported to be lethal in sec24-1. However, here we constructed a sec24-1 Deltasfb3 and a sec24-1 Deltasfb2 Deltasfb3 strain and show that Hsp150 was secreted slowly in both. Turning off the SEC24 gene did not inhibit Hsp150 secretion either, and the lack of SEC24 expression in a Deltasfb2 Deltasfb3 deletant still allowed some secretion. The sec24-1 Deltasfb2 Deltasfb3 mutant grew slower than sec24-1. The cells were irregularly shaped, budded from random sites and contained proliferated ER at permissive temperature. At restrictive temperature, the ER formed carmellae-like proliferations. Our data indicate that ER exit may occur in vesicles lacking a full complement of Sec23p/24p and Sec13p/31p, demonstrating diversity in the composition of the COPII coat.     

New Insights into the Structural Mechanisms of the COPII Coat

In eukaryotes, coat protein complex II (COPII) proteins are involved in transporting cargo proteins from the endoplasmic reticulum (ER) to the Golgi apparatus. The COPII proteins, Sar1, Sec23/24, and Sec13/31 polymerize into a coat that gathers cargo proteins into a coated vesicle. Structures have been recently solved of individual COPII proteins, COPII proteins in complex with cargo, and higher‐order COPII coat assemblies. In this review, we will summarize the latest developments in COPII structure and discuss how these structures shed light on the functional mechanisms of the COPII coat.     

Sec24p and Sec16p cooperate to regulate the GTP cycle of the COPII coat

Vesicle budding from the endoplasmic reticulum (ER) employs a cycle of GTP binding and hydrolysis to regulate assembly of the COPII coat. We have identified a novel mutation (sec24-m11) in the cargo-binding subunit, Sec24p, that specifically impacts the GTP-dependent generation of vesicles in vitro. Using a high-throughput approach, we defined genetic interactions between sec24-m11 and a variety of trafficking components of the early secretory pathway, including the candidate COPII regulators, Sed4p and Sec16p. We defined a fragment of Sec16p that markedly inhibits the Sec23p- and Sec31p-stimulated GTPase activity of Sar1p, and demonstrated that the Sec24p-m11 mutation diminished this inhibitory activity, likely by perturbing the interaction of Sec24p with Sec16p. The consequence of the heightened GTPase activity when Sec24p-m11 is present is the generation of smaller vesicles, leading to accumulation of ER membranes and more stable ER exit sites. We propose that association of Sec24p with Sec16p creates a novel regulatory complex that retards the GTPase activity of the COPII coat to prevent premature vesicle scission, pointing to a fundamental role for GTP hydrolysis in vesicle release rather than in coat assembly/disassembly.     

Sec16p potentiates the action of COPII proteins to bud transport vesicles

Listeria monocytogenes is a facultative intracellular bacterial pathogen that escapes from a phagosome and grows in the host cell cytosol. The pore-forming cholesterol-dependent cytolysin, listeriolysin O (LLO), mediates bacterial escape from vesicles and is approximately 10-fold more active at an acidic than neutral pH. By swapping dissimilar residues from a pH-insensitive orthologue, perfringolysin O (PFO), we identified leucine 461 as unique to pathogenic Listeria and responsible for the acidic pH optimum of LLO. Conversion of leucine 461 to the threonine present in PFO increased the hemolytic activity of LLO almost 10-fold at a neutral pH. L. monocytogenes synthesizing LLO L461T, expressed from its endogenous site on the bacterial chromosome, resulted in a 100-fold virulence defect in the mouse listeriosis model. These bacteria escaped from acidic phagosomes and initially grew normally in cells and spread cell to cell, but prematurely permeabilized the host membrane and killed the cell. These data show that the acidic pH optimum of LLO results from an adaptive mutation that acts to limit cytolytic activity to acidic vesicles and prevent damage in the host cytosol, a strategy also used by host cells to compartmentalize lysosomal hydrolases.     

In vivo trafficking and localization of p24 proteins in plant cells

p24 proteins constitute a family of putative cargo receptors that traffic in the early secretory pathway. p24 proteins can be divided into four subfamilies (p23, p24, p25 and p26) by sequence homology. In contrast to mammals and yeast, most plant p24 proteins contain in their cytosolic C-terminus both a dilysine motif in the −3, −4 position and a diaromatic motif in the −7, −8 position. We have previously shown that the cytosolic tail of Arabidopsis p24 proteins has the ability to interact with ARF1 and coatomer (through the dilysine motif) and with COPII subunits (through the diaromatic motif). Here, we establish the localization and trafficking properties of an Arabidopsis thaliana p24 protein ( At p24) and have investigated the contribution of the sorting motifs in its cytosolic tail to its in vivo localization. At p24-red fluorescent protein localizes exclusively to the endoplasmic reticulum (ER), in contrast with the localization of p24 proteins in other eukaryotes, and the dilysine motif is necessary and sufficient for ER localization. In contrast, At p24 mutants lacking the dilysine motif are transported along the secretory pathway to the prevacuolar compartment and the vacuole, although a significant fraction is also found at the plasma membrane. Finally, we have found that ER export of At p24 is COPII dependent, while its ER localization requires COPI function, presumably for efficient Golgi to ER recycling.     

Scribble-mediated membrane targeting of PHLPP1 is required for its negative regulation of Akt

PHLPP1 (PH domain leucine-rich-repeats protein phosphatase) is a Ser/Thr protein phosphatase that acts as a tumour suppressor by negatively regulating Akt. Here, we show that PHLPP1 is recruited to the cell membrane by binding to a scaffolding protein: Scribble. Knockdown of Scribble (Scrib) results in redistribution of PHLPP1 from the membrane to the cytoplasm and an increase in Akt phosphorylation, whereas overexpression of Scrib has the opposite effect. Furthermore, PHLPP1-dependent inhibition of cell proliferation is facilitated by the formation of a Scrib, PHLPP1 and Akt trimeric complex. Thus, our findings identify a functional interaction between PHLPP1 and Scrib in negatively regulating Akt signalling.     

The synthetic peptide RPRAATF allows specific assay of Akt activity in cell lysates

The Akt protein kinase is a critical signaling molecule in a range of cellular processes. A key to identifying the role of this pleiotropic kinase in any particular process is the ability to quantitate its activity. In this study we show that the synthetic peptide RPRAATF is a specific substrate for the kinase in crude cell extracts, thus enabling rapid, convenient, and sensitive assay of Akt activity. Peptide kinase activity was confined to a single peak upon sequential ion-exchange chromatography of whole-cell extracts of Balb/c 3T3 fibroblasts. This activity was stimulated by both platelet-derived growth factor and pervanadate, phosphatidyl inositol 3-kinase dependent, and inhibited by specific immunodepletion with anti-Akt antisera. Furthermore, direct assays of crude extracts from a range of cell types using this peptide were consistent with the results obtained using specific immunoprecipitation assays.     

Phosphorylation of Akt at the C-terminal tail triggers Akt Activation

Aberrant hyper-activation of the protein kinase Akt plays a critical role in promoting tumorigenesis. Mechanistically, previous studies establish that phosphorylation of Akt at S473 and T308 by mTORC2 and PDK1, respectively, is necessary for its full activation, thereby having been used as Akt activation markers. Recently, we report that phosphorylation of S477 and T479 at the extreme C-terminus of Akt1 promotes Akt1 activation. We further demonstrate that Akt1 pS477 and pT479 events are governed by Cdk2/Cyclin A or mTORC2 under distinct cellular contexts such as cell cycle progression or growth stimulation conditions. Here, we summarize our major findings regarding the biological significance for pS477/pT479-mediated activation of Akt and also provide perspectives for future follow-up studies.     

Receptor-mediated ER export of human MHC class I molecules is regulated by the C-terminal single amino acid

Major histocompatibility complex class I (MHC-I) molecules bind antigens in the endoplasmic reticulum (ER) and deliver them to the cell surface for immune surveillance of viruses and tumors. Whereas key steps of MHC-I assembly and its acquisition of peptides in the ER are relatively well defined, little is known about how MHC-I molecules leave the ER for cell surface expression. Here, we show that ER export of human classical MHC-I molecules (HLA-A/-B/-C) is regulated by their C-terminal single amino acid, valine or alanine. These amino acids, conserved in nearly all known human MHC-I alleles, serve as the ER export signal by binding to the Sec23/24 complex, a structural component of coat protein complex II (COPII) vesicles that mediate ER-to-Golgi trafficking. Together, our results strongly suggest that ER export of human classical MHC-I molecules can occur via a receptor-mediated process dictated by a highly conserved ER export signal.     

Proteomic Profiling of Mammalian COPII and COPI Vesicles

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Two novel effectors of trafficking and maturation of the yeast plasma membrane H+‐ATPase

The endoplasmic reticulum (ER) is the entry site of proteins into the endomembrane system. Proteins exit the ER via coat protein II (COPII) vesicles in a selective manner, mediated either by direct interaction with the COPII coat or aided by cargo receptors. Despite the fundamental role of such receptors in protein sorting, only a few have been identified. To further define the machinery that packages secretory cargo and targets proteins from the ER to Golgi membranes, we used multiple systematic approaches, which revealed 2 uncharacterized proteins that mediate the trafficking and maturation of Pma1, the essential yeast plasma membrane proton ATPase. Ydl121c (Exp1) is an ER protein that binds Pma1, is packaged into COPII vesicles, and whose deletion causes ER retention of Pma1. Ykl077w (Psg1) physically interacts with Exp1 and can be found in the Golgi and coat protein I (COPI) vesicles but does not directly bind Pma1. Loss of Psg1 causes enhanced degradation of Pma1 in the vacuole. Our findings suggest that Exp1 is a Pma1 cargo receptor and that Psg1 aids Pma1 maturation in the Golgi or affects its retrieval. More generally our work shows the utility of high content screens in the identification of novel trafficking components.      

sec24d encoding a component of COPII is essential for vertebra formation, revealed by the analysis of the medaka mutant, vbi

We characterized a medaka mutant, vertebra imperfecta (vbi), that displays skeletal defects such as craniofacial malformation and delay of vertebra formation. Positional cloning analysis revealed a nonsense mutation in sec24d encoding a component of the COPII coat that plays a role in anterograde protein trafficking from the endoplasmic reticulum (ER) to the Golgi apparatus. Immunofluorescence analysis revealed the accumulation of type II collagen in the cytoplasm of craniofacial chondrocytes, notochord cells, and the cells on the myoseptal boundary in vbi mutants. Electron microscopy analysis revealed dilation of the ER and defective secretion of ECM components from cells in both the craniofacial cartilage and notochord in vbi. The higher vertebrates have at least 4 sec24 paralogs; however, the function of each paralog in development remains unknown. sec24d is highly expressed in the tissues that are rich in extracellular matrix and is essential for the secretion of ECM component molecules leading to the formation of craniofacial cartilage and vertebra.     

24p3-24p3受体系统：一种新的铁转运途径

小鼠24p3是脂质运载蛋白lipocalin家族的成员之一,可在白介素3(interleukin-3,IL-3)缺乏时诱导细胞发生凋亡,并参与细胞的铁转运过程.最近,Devireddy等又成功克隆到了24p3的细胞表面受体(24p3 receptor,24p3R),进一步确定了24p3-24p3R这一新的铁转运途径.该途径的发现不仅增加了对铁转运理论新的认识,更重要的是,这一发现为铁代谢调控细胞凋亡理论的建立奠定了基础.     

Alterations in microRNA expression profile in HCV-infected hepatoma cells: Involvement of miR-491 in regulation of HCV replication via the PI3 kinase/Akt pathway

The aim of this study was to investigate the role of microRNA (miRNA) on hepatitis C virus (HCV) replication in hepatoma cells. Using miRNA array analysis, miR-192/miR-215, miR-194, miR-320, and miR-491 were identified as miRNAs whose expression levels were altered by HCV infection. Among them, miR-192/miR-215 and miR-491 were capable of enhancing replication of the HCV replicon as well as HCV itself. HCV IRES activity or cell proliferation was not increased by forced expression of miR-192/miR-215 or miR-491. Investigation of signaling pathways revealed that miR-491 specifically suppressed the phosphoinositol-3 (PI3) kinase/Akt pathway. Under inhibition of PI3 kinase by LY294002, the suppressive effect of miR-491 on HCV replication was abolished, indicating that suppression of HCV replication by miR-491 was dependent on the PI3 kinase/Akt pathway. miRNAs altered by HCV infection would then affect HCV replication, which implies a complicated mechanism for regulating HCV replication. HCV-induced miRNA may be involved in changes in cellular properties including hepatocarcinogenesis.