AGD5 is a GTPase-activating protein at the trans-Golgi network

ARF‐GTPases are important proteins that control membrane trafficking events. Their activity is largely influenced by the interplay between guanine nucleotide exchange factors (GEFs) and GTPase‐activating proteins (GAPs), which facilitate the activation or inactivation of ARF‐GTPases, respectively. There are 15 predicted proteins that contain an ARF‐GAP domain within the Arabidopsis thaliana genome, and these are classified as ARF‐GAP domain (AGD) proteins. The function and subcellular distribution of AGDs, including the ability to activate ARF‐GTPases in vivo, that remain largely uncharacterized to date. Here we show that AGD5 is localised to the trans‐Golgi network (TGN), where it co‐localises with ARF1, a crucial GTPase that is involved in membrane trafficking and which was previously shown to be distributed on Golgi and post‐Golgi structures of unknown nature. Taking advantage of the in vivo AGD5–ARF1 interaction at the TGN, we show that mutation of an arginine residue that is critical for ARF‐GAP activity of AGD5 leads to longer residence of ARF1 on the membranes, as expected if GTP hydrolysis on ARF1 was impaired due to a defective GAP. Our results establish the nature of the post‐Golgi compartments in which ARF1 localises, as well as identifying the role of AGD5 in vivo as a TGN‐localised GAP. Furthermore, in vitro experiments established the promiscuous interaction between AGD5 and the plasma membrane‐localised ADP ribosylation factor B (ARFB), confirming that ARF‐GAP specificity for ARF‐GTPases within the cell environment may be spatially regulated.     

Pilt is a coiled-coil domain-containing protein that localizes at the trans-Golgi complex and regulates its structure

Protein incorporated later into tight junctions (Pilt), also termed tight junction-associated protein 1 or tight junction protein 4, is a coiled-coil domain-containing protein that was originally identified as a human discs large-interacting protein. In this study, we identified Pilt as an Arf6-binding protein by yeast two-hybrid screening. By immunocytochemical analysis, Pilt was shown to be predominantly localized at the trans-Golgi complex and to exhibit diffuse cytoplasmic distribution in association with endosomes and plasma membrane in NIH3T3 cells. Silencing of endogenous Pilt disrupted the Golgi structure. The present findings suggest the functional involvement of Pilt in the maintenance of the Golgi structure.

Structured summary of protein interactions

GM130 and Piltcolocalize by fluorescence microscopy (View interaction)Arf6(Q67L)physically interacts with Pilt by two hybrid (View Interaction: 1, 2)Piltphysically interacts with Arf6(Q67L) by pull down (View interaction)     

Lowe syndrome protein OCRL1 interacts with clathrin and regulates protein trafficking between endosomes and the trans-Golgi network

Oculocerebrorenal syndrome of Lowe is caused by mutation of OCRL1, a phosphatidylinositol 4,5-bisphosphate 5-phosphatase localized at the Golgi apparatus. The cellular role of OCRL1 is unknown, and consequently the mechanism by which loss of OCRL1 function leads to disease is ill defined. Here, we show that OCRL1 is associated with clathrin-coated transport intermediates operating between the trans-Golgi network (TGN) and endosomes. OCRL1 interacts directly with clathrin heavy chain and promotes clathrin assembly in vitro. Interaction with clathrin is not, however, required for membrane association of OCRL1. Overexpression of OCRL1 results in redistribution of clathrin and the cation-independent mannose 6-phosphate receptor (CI-MPR) to enlarged endosomal structures that are defective in retrograde trafficking to the TGN. Depletion of cellular OCRL1 also causes partial redistribution of a CI-MPR reporter to early endosomes. These findings suggest a role for OCRL1 in clathrin-mediated trafficking of proteins from endosomes to the TGN and that defects in this pathway might contribute to the Lowe syndrome phenotype.     

A prostate-derived cDNA that is mapped to human chromosome 19 encodes a novel protein

The epithelial cells of prostate gland secrete various secretory products that play an important role in the growth and differentiation of prostate gland. These secretory products have also been implicated in neuroendocrine differentiation of benign prostatic hyperplasia and prostate malignancy. We have cloned a prostate-derived cDNA encoding a novel protein with a predicted molecular weight of 78 kDa (P(78)), and precisely mapped the cDNA sequence to chromosome 19. The P(78) gene has a complex genomic structure with 18 exons and 17 introns. The P(78) contains two conserved structural domains with limited similarity to domain D of synapsin I. The P(78) mRNA was expressed in various human cell lines. Western blot analysis using antibody specific for the P(78) revealed the presence of the P(78) protein in the prostate cancer cell lines with much lower level in metastatic prostate cancer cell lines compared to that in a primary prostate cancer cell line.     

BAP,a mammalian BiP-associated protein,is a nucleotide exchange factor that regulates the ATPase activity of BiP

We identified a mammalian BiP-associated protein, BAP, using a yeast two-hybrid screen that shared low homology with yeast Sls1p/Sil1p and mammalian HspBP1, both of which regulate the ATPase activity of their Hsp70 partner. BAP encoded an approximately 54-kDa protein with an N-terminal endoplasmic reticulum (ER) targeting sequence, two sites of N-linked glycosylation, and a C-terminal ER retention sequence. Immunofluorescence staining demonstrated that BAP co-localized with GRP94 in the endoplasmic reticulum. BAP was ubiquitously expressed but showed the highest levels of expression in secretory organ tissues, a pattern similar to that observed with BiP. BAP binding was affected by the conformation of the ATPase domain of BiP based on in vivo binding studies with BiP mutants. BAP stimulated the ATPase activity of BiP when added alone or together with the ER DnaJ protein, ERdj4, by promoting the release of ADP from BiP. Together, these data demonstrate that BAP serves as a nucleotide exchange factor for BiP and provide insights into the mechanisms that control protein folding in the mammalian ER.     

A novel clathrin homolog that co-distributes with cytoskeletal components functions in the trans-Golgi network

A clathrin homolog encoded on human chromosome 22 (CHC22) displays distinct biochemistry, distribution and function compared with conventional clathrin heavy chain (CHC17), encoded on chromosome 17. CHC22 protein is upregulated during myoblast differentiation into myotubes and is expressed at high levels in muscle and at low levels in non-muscle cells, relative to CHC17. The trimeric CHC22 protein does not interact with clathrin heavy chain subunits nor bind significantly to clathrin light chains. CHC22 associates with the AP1 and AP3 adaptor complexes but not with AP2. In non-muscle cells, CHC22 localizes to perinuclear vesicular structures, the majority of which are not clathrin coated. Treatments that disrupt the actin-myosin cytoskeleton or affect sorting in the trans-Golgi network (TGN) cause CHC22 redistribution. Overexpression of a subdomain of CHC22 induces altered distribution of TGN markers. Together these results implicate CHC22 in TGN membrane traffic involving the cytoskeleton.     

RBM33 is a unique m6A RNA-binding protein that regulates ALKBH5 demethylase activity and substrate selectivity

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A novel growth-inducible gene that encodes a protein with a conserved cold-shock domain.

We have isolated a cDNA that encodes a novel member of the Y-box binding protein family, termed as RYB-a (Rat Y-box Binding protein-a). RYB-a is a 31 kDa protein that contains a conserved cold-shock domain and an amino acid alignment similar to those of charge zipper proteins. Expression of RYB-a mRNA was highly abundant in the skeletal muscle, spleen, and fetal liver. The expression is very low in new-born and adult livers, suggesting its expression is under developmental regulation. In addition, the expression of RYB-a mRNA was induced in the liver during regeneration and by stimulation of quiescent fibroblast cells with serum. Induction in the fibroblasts was inhibited by treating the cell with a specific tyrosine kinase inhibitor, genistein or by detachment of cell-adhesion. Since both treatments are known to inhibit G1 cells to enter S phase, RYB-a gene is thought to be a member of growth-inducible genes.     

cGMP-dependent protein kinase I gamma encodes a nuclear localization signal that regulates nuclear compartmentation and function

cGMP-dependent protein kinase I (PKGI) plays an important role in regulating how cGMP specifies vascular smooth muscle cell (SMC) phenotype. Although studies indicate that PKGI nuclear localization controls how cGMP regulates gene expression in SMC, information about the mechanisms that regulate PKGI nuclear compartmentation and its role in directly regulating cell phenotype is limited. Here we characterize a nuclear localization signal sequence (NLS) in PKGIγ, a proteolytically cleaved PKGI kinase fragment that translocates to the nucleus of SMC. Immuno-localization studies using cells expressing native and NLS-mutant PKGIγ, and treated with a small molecule nuclear transport inhibitor, indicated that PKGIγ encodes a constitutively active NLS that requires importin α and β for regulation of its compartmentation. Moreover, studies utilizing a genetically encoded nuclear phospho-CREB biosensor probe and fluorescence lifetime imaging microscopy demonstrated that this NLS controls PKGIγ nuclear function. In addition, although cytosolic PKGIγ-activity was observed to stimulate MAPK/ERK-mediated nuclear CREB signaling in SMC, NLS-mediated PKGIγ nuclear activity alone was determined to increase the expression of differentiation marker proteins in these cells. These results indicate that NLS-mediated nuclear PKGIγ localization plays an important role in how PKGI regulates vascular SMC phenotype.     

Ccpg1, a novel scaffold protein that regulates the activity of the Rho guanine nucleotide exchange factor Dbs

Dbs is a Rho-specific guanine nucleotide exchange factor (RhoGEF) with in vitro exchange activity specific for RhoA and Cdc42. Like many RhoGEF family members, the in vivo exchange activity of Dbs is restricted in a cell-specific manner. Here we report the characterization of a novel scaffold protein (designated cell cycle progression protein 1 [Ccpg1]) that interacts with Dbs and modulates its in vivo exchange specificity. When coexpressed in mammalian cells, Ccpg1 binds to the Dbl homology/pleckstrin homology domain tandem motif of Dbs and inhibits its exchange activity toward RhoA, but not Cdc42. Expression of Ccpg1 correlates with the ability of Dbs to activate endogenous RhoA in cultured cells, and suppression of endogenous Ccpg1 expression potentiates Dbs exchange activity toward RhoA. The isolated Dbs binding domain of Ccpg1 is not sufficient to suppress Dbs exchange activity on RhoA, thus suggesting a regulatory interaction. Ccpg1 mediates recruitment of endogenous Src kinase into Dbs-containing complexes and interacts with the Rho family member Cdc42. Collectively, our studies suggest that Ccpg1 represents a new class of regulatory scaffold protein that can function as both an assembly platform for Rho protein signaling complexes and a regulatory protein which can restrict the substrate utilization of a promiscuous RhoGEF family member.     

A Highlights from MBoC Selection: Phosphatidylserine translocation at the yeast trans-Golgi network regulates protein sorting into exocytic vesicles

Sorting of plasma membrane proteins into exocytic vesicles at the yeast trans-Golgi network (TGN) is believed to be mediated by their coalescence with specific lipids, but how these membrane-remodeling events are regulated is poorly understood. Here we show that the ATP-dependent phospholipid flippase Drs2 is required for efficient segregation of cargo into exocytic vesicles. The plasma membrane proteins Pma1 and Can1 are missorted from the TGN to the vacuole in drs2∆ cells. We also used a combination of flippase mutants that either gain or lose the ability to flip phosphatidylserine (PS) to determine that PS flip by Drs2 is its critical function in this sorting event. The primary role of PS flip at the TGN appears to be to control the oxysterol-binding protein homologue Kes1/Osh4 and regulate ergosterol subcellular distribution. Deletion of KES1 suppresses plasma membrane–missorting defects and the accumulation of intracellular ergosterol in drs2 mutants. We propose that PS flip is part of a homeostatic mechanism that controls sterol loading and lateral segregation of protein and lipid domains at the TGN.     

A human placental cDNA clone that encodes nonhistone chromosomal protein HMG-1.

From a human placental lambda gt11 cDNA library, we have isolated a cDNA clone that encodes the entire 215-residue amino acid sequence of HMG-1. Analysis of an internal sequence similarity suggests that the DNA-binding domains of HMG-1 are separated by a rather long and flexible linker segment. Southern blotting of DNA digested with BamHI indicated a highly variable number of genes (or pseudogenes) for HMG-1 in different species. Characterization of HMG-1 mRNA expression by Northern blotting showed that three mRNA species of approximately 1.0, 1.4 and 2.4 kb were expressed in all mammalian organs and cell lines examined. These included several rat organs at different stages of development. Northern analysis also suggested the occurrence of HMG-1 mRNA in an invertebrate and a plant species.     

Hephaestus encodes a polypyrimidine tract binding protein that regulates Notch signalling during wing development in Drosophila melanogaster

We describe the role of the Drosophila melanogaster hephaestus gene in wing development. We have identified several hephaestus mutations that map to a gene encoding a predicted RNA-binding protein highly related to human polypyrimidine tract binding protein and Xenopus laevis 60 kDa Vg1 mRNA-binding protein. Polypyrimidine tract binding proteins play diverse roles in RNA processing including the subcellular localization of mRNAs, translational control, internal ribosome entry site use, and the regulation of alternate exon selection. The analysis of gene expression in imaginal discs and adult cuticle of genetic mosaic animals supports a role for hephaestus in Notch signalling. Somatic clones lacking hephaestus express the Notch target genes wingless and cut, induce ectopic wing margin in adjacent wild-type tissue, inhibit wing-vein formation and have increased levels of Notch intracellular domain immunoreactivity. Clones mutant for both Delta and hephaestus have the characteristic loss-of-function thick vein phenotype of DELTA: These results lead to the hypothesis that hephaestus is required to attenuate Notch activity following its activation by Delta. This is the first genetic analysis of polypyrimidine tract binding protein function in any organism and the first evidence that such proteins may be involved in the Notch signalling pathway.     

Notchless encodes a novel WD40-repeat-containing protein that modulates Notch signaling activity.

Signaling by Notch family receptors is involved in many cell-fate decisions during development. Several modifiers of Notch activity have been identified, suggesting that regulation of Notch signaling is complex. In a genetic screen for modifiers of Notch activity, we identified a gene encoding a novel WD40-repeat protein. The gene is called Notchless, because loss-of-function mutant alleles dominantly suppress the wing notching caused by certain Notch alleles. Reducing Notchless activity increases Notch activity. Overexpression of Notchless in Xenopus or Drosophila appears to have a dominant-negative effect in that it also increases Notch activity. Biochemical studies show that Notchless binds to the cytoplasmic domain of Notch, suggesting that it serves as a direct regulator of Notch signaling activity.