scholarly journals Mistargeting of the Lectin ERGIC-53 to the Endoplasmic Reticulum of HeLa Cells Impairs the Secretion of a Lysosomal Enzyme

1998 ◽  
Vol 142 (2) ◽  
pp. 377-389 ◽  
Author(s):  
Florence Vollenweider ◽  
Felix Kappeler ◽  
Christian Itin ◽  
Hans-Peter Hauri
Keyword(s):  
Hela Cells ◽  
Lysosomal Enzyme ◽  
Intracellular Transport ◽  
Secretory Pathway ◽  
Small Subset ◽  
Mutant Form ◽  
Membrane Glycoproteins ◽  
Early Secretory Pathway ◽  
Glycoprotein Secretion

ERGIC-53, a homo-oligomeric recycling protein associated with the ER–Golgi intermediate compartment (ERGIC), has properties of a mannose-selective lectin in vitro, suggesting that it may function as a transport receptor for glycoproteins in the early secretory pathway. To investigate if ERGIC-53 is involved in glycoprotein secretion, a mutant form of this protein was generated that is incapable of leaving the ER. If expressed in HeLa cells in a tetracycline-inducible manner, this mutant accumulated in the ER and retained the endogenous ERGIC-53 in this compartment, thus preventing its recycling. Mistargeting of ERGIC-53 to the ER did not alter the gross morphology of the early secretory pathway, including the distribution of β′-COP. However, it impaired the secretion of one major glycoprotein, identified as the precursor of the lysosomal enzyme cathepsin C, while overexpression of wild-type ERGIC-53 had no effect on glycoprotein secretion. Transport of two other lysosomal enzymes and three post-Golgi membrane glycoproteins was unaffected by inactivating the recycling of ERGIC-53. The results suggest that the recycling of ERGIC-53 is required for efficient intracellular transport of a small subset of glycoproteins, but it does not appear to be essential for the majority of glycoproteins.

scholarly journals Casein Kinase I Regulates Membrane Binding by ARF GAP1

2002 ◽  
Vol 13 (8) ◽  
pp. 2559-2570 ◽  
Author(s):  
Sidney Yu ◽  
Michael G. Roth
Keyword(s):  
Amino Acid ◽  
Secretory Pathway ◽  
Protein Transport ◽  
Membrane Binding ◽  
Casein Kinase ◽  
Protein Domain ◽  
Amino Terminal ◽  
Early Secretory Pathway

ARF GAP1, a 415-amino acid GTPase activating protein (GAP) for ADP-ribosylation factor (ARF) contains an amino-terminal 115-amino acid catalytic domain and no other recognizable features. Amino acids 203–334 of ARF GAP1 were sufficient to target a GFP-fusion protein to Golgi membranes in vivo. When overexpressed in COS-1 cells, this protein domain inhibited protein transport between the ER and Golgi and, in vitro, competed with the full-length ARF GAP1 for binding to membranes. Membrane binding by ARF GAP1 in vitro was increased by a factor in cytosol and this increase was inhibited by IC261, an inhibitor selective for casein kinase Iδ (CKIδ), or when cytosol was treated with antibody to CKIδ. The noncatalytic domain of ARF GAP1 was phosphorylated both in vivo and in vitro by CKI. IC261 blocked membrane binding by ARF GAP1 in vivo and inhibited protein transport in the early secretory pathway. Overexpression of a catalytically inactive CKIδ also inhibited the binding of ARF GAP1 to membranes and interfered with protein transport. Thus, a CKI isoform is required for protein traffic through the early secretory pathway and can modulate the amount of ARF GAP1 that can bind to membranes.

scholarly journals Early secretory pathway localization and lack of processing for hepatitis E virus replication protein pORF1

2013 ◽  
Vol 94 (4) ◽  
pp. 807-816 ◽  
Author(s):  
Julia Perttilä ◽  
Pirjo Spuul ◽  
Tero Ahola
Keyword(s):  
Hepatitis E Virus ◽  
Secretory Pathway ◽  
Hepatitis E ◽  
Proteolytic Processing ◽  
Full Length ◽  
Replication Protein ◽  
Positive Strand ◽  
Early Secretory Pathway ◽  
Positive Strand Rna

Hepatitis E virus (HEV) is a positive-strand RNA virus and a major causative agent of acute sporadic and epidemic hepatitis. HEV replication protein is encoded by ORF1 and contains the predicted domains of methyltransferase (MT), protease, macro domain, helicase (HEL) and polymerase (POL). In this study, the full-length protein pORF1 (1693 aa) and six truncated variants were expressed by in vitro translation and in human HeLa and hepatic Huh-7 cells by using several vector systems. The proteins were visualized by three specific antisera directed against the MT, HEL and POL domains. In vitro translation of full-length pORF1 yielded smaller quantities of two fragments. However, these fragments were not observed after pORF1 expression and pulse–chase studies in human cells, and their production was not dependent on the predicted protease domain in pORF1. The weight of evidence supports the proposition that pORF1 is not subjected to specific proteolytic processing, which is unusual among animal positive-strand RNA viruses but common for plant viruses. pORF1 was membrane associated in cells and localized to a perinuclear region, where it partially overlapped with localization of the endoplasmic reticulum (ER) marker BAP31 and was closely interspersed with staining of the ER–Golgi intermediate compartment marker protein ERGIC-53. Co-localization with BAP31 was enhanced by treatment with brefeldin A. Therefore, HEV may utilize modified early secretory pathway membranes for replication.

scholarly journals A Novel Group of Glutaredoxins in the cis-Golgi Critical for Oxidative Stress Resistance

2008 ◽  
Vol 19 (6) ◽  
pp. 2673-2680 ◽  
Author(s):  
Nikola Mesecke ◽  
Anne Spang ◽  
Marcel Deponte ◽  
Johannes M. Herrmann
Keyword(s):  
Disulfide Bonds ◽  
Secretory Pathway ◽  
Reduced Glutathione ◽  
General Role ◽  
Growth Defects ◽  
Early Secretory Pathway ◽  
Full Complement ◽  
Increased Sensitivity ◽  
Substrate Proteins

Glutaredoxins represent a ubiquitous family of proteins that catalyze the reduction of disulfide bonds in their substrate proteins by use of reduced glutathione. In an attempt to identify the full complement of glutaredoxins in baker's yeast, we found three so-far uncharacterized glutaredoxin-like proteins that we named Grx6, Grx7, and Grx8. Grx6 and Grx7 represent closely related monothiol glutaredoxins that are synthesized with N-terminal signal sequences. Both proteins are located in the cis-Golgi, thereby representing the first glutaredoxins found in a compartment of the secretory pathway. In contrast to formerly described monothiol glutaredoxins, Grx6 and Grx7, showed a high glutaredoxin activity in vitro. Grx6 and Grx7 overlap in their activity and deletion mutants lacking both proteins show growth defects and a strongly increased sensitivity toward oxidizing agents such as hydrogen peroxide or diamide. Our observations suggest that Grx6 and Grx7 do not play a general role in the oxidative folding of proteins in the early secretory pathway but rather counteract the oxidation of specific thiol groups in substrate proteins.

scholarly journals The N-terminal domain of the human eIF2β subunit and the CK2 phosphorylation sites are required for its function

2006 ◽  
Vol 394 (1) ◽  
pp. 227-236 ◽  
Author(s):  
Franc Llorens ◽  
Anna Duarri ◽  
Eduard Sarró ◽  
Nerea Roher ◽  
Maria Plana ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Hela Cells ◽  
Mutant Cell ◽  
Phosphorylation Site ◽  
Translation Initiation Factor ◽  
Initiation Factor ◽  
Mutant Form ◽  
Wild Type ◽  
Main Site

CK2 (protein kinase CK2) is known to phosphorylate eIF2 (eukaryotic translation initiation factor 2) in vitro; however, its implication in this process in living cells has remained to be confirmed. The combined use of chemical inhibitors (emodin and apigenin) of CK2 together with transfection experiments with the wild-type of the K68A kinase-dead mutant form of CK2α evidenced the direct involvement of this protein kinase in eIF2β phosphorylation in cultured HeLa cells. Transfection of HeLa cells with human wild-type eIF2β or its phosphorylation site mutants showed Ser2 as the main site for constitutive eIF2β phosphorylation, whereas phosphorylation at Ser67 seems more restricted. In vitro phosphorylation of eIF2β also pointed to Ser2 as a preferred site for CK2 phosphorylation. Overexpression of the eIF2β S2/67A mutant slowed down the rate of protein synthesis stimulated by serum, although less markedly than the overexpression of the Δ2–138 N-terminal-truncated form of eIF2β (eIF2β-CT). Mutation at Ser2 and Ser67 did not affect eIF2β integrating into the eIF2 trimer or being able to complex with eIF5 and CK2α. The eIF2β-CT form was also incorporated into the eIF2 trimer but did not bind to eIF5. Overexpression of eIF2β slightly decreased HeLa cell viability, an effect that was more evident when overexpressing the eIF2β S2/67A mutant. Cell death was particularly marked when overexpressing the eIF2β-CT form, being detectable at doses where eIF2β and eIF2β S2/67A were ineffective. These results suggest that Ser2 and Ser67 contribute to the important role of the N-terminal region of eIF2β for its function in mammals.

scholarly journals A Novel Cellular Protein, VPEF, Facilitates Vaccinia Virus Penetration into HeLa Cells through Fluid Phase Endocytosis

2008 ◽  
Vol 82 (16) ◽  
pp. 7988-7999 ◽  
Author(s):  
Cheng-Yen Huang ◽  
Tsai-Yi Lu ◽  
Chi-Horng Bair ◽  
Yuan-Shau Chang ◽  
Jeng-Kuan Jwo ◽  
...  
Keyword(s):  
Vaccinia Virus ◽  
Hela Cells ◽  
Intracellular Transport ◽  
Fluid Phase ◽  
Cellular Protein ◽  
Cell Entry ◽  
Surface Lipid ◽  
Fluid Phase Endocytosis

ABSTRACT Vaccinia virus is a large DNA virus that infects many cell cultures in vitro and animal species in vivo. Although it has been used widely as a vaccine, its cell entry pathway remains unclear. In this study, we showed that vaccinia virus intracellular mature virions bound to the filopodia of HeLa cells and moved toward the cell body and entered the cell through an endocytic route that required a dynamin-mediated pathway but not a clathrin- or caveola-mediated pathway. Moreover, virus penetration required a novel cellular protein, vaccinia virus penetration factor (VPEF). VPEF was detected on cell surface lipid rafts and on vesicle-like structures in the cytoplasm. Both vaccinia virus and dextran transiently colocalized with VPEF, and, importantly, knockdown of VPEF expression blocked vaccinia virus penetration as well as intracellular transport of dextran, suggesting that VPEF mediates vaccinia virus entry through a fluid uptake endocytosis process in HeLa cells. Intracellular VPEF-containing vesicles did not colocalize with Rab5a or caveolin but partially colocalized with Rab11, supporting the idea that VPEF plays a role in vesicle trafficking and recycling in HeLa cells. In summary, this study characterized the mechanism by which vaccinia virus enters HeLa cells and identified a cellular factor, VPEF, that is exploited by vaccinia virus for cell entry through fluid phase endocytosis.

scholarly journals HtrA2 Regulates β-Amyloid Precursor Protein (APP) Metabolism through Endoplasmic Reticulum-associated Degradation

2007 ◽  
Vol 282 (38) ◽  
pp. 28285-28295 ◽  
Author(s):  
Henri J. Huttunen ◽  
Suzanne Y. Guénette ◽  
Camilla Peach ◽  
Christopher Greco ◽  
Weiming Xia ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Secretory Pathway ◽  
Proteasome Inhibition ◽  
Precursor Protein ◽  
Amyloid Peptide ◽  
Early Secretory Pathway ◽  
App Metabolism ◽  
Microsomal Membranes ◽  
Β Amyloid

Alzheimer disease-associated β-amyloid peptide is generated from its precursor protein APP. By using the yeast two-hybrid assay, here we identified HtrA2/Omi, a stress-responsive chaperone-protease as a protein binding to the N-terminal cysteinerich region of APP. HtrA2 coimmunoprecipitates exclusively with immature APP from cell lysates as well as mouse brain extracts and degrades APP in vitro. A subpopulation of HtrA2 localizes to the cytosolic side of the endoplasmic reticulum (ER) membrane where it contributes to ER-associated degradation of APP together with the proteasome. Inhibition of the proteasome results in accumulation of retrotranslocated forms of APP and increased association of APP with HtrA2 and Derlin-1 in microsomal membranes. In cells lacking HtrA2, APP holoprotein is stabilized and accumulates in the early secretory pathway correlating with elevated levels of APP C-terminal fragments and increased Aβ secretion. Inhibition of ER-associated degradation (either HtrA2 or proteasome) promotes binding of APP to the COPII protein Sec23 suggesting enhanced trafficking of APP out of the ER. Based on these results we suggest a novel function for HtrA2 as a regulator of APP metabolism through ER-associated degradation.

Membrane insertion and intracellular transport of yeast syntaxin Sso2p in mammalian cells

1994 ◽  
Vol 107 (12) ◽  
pp. 3623-3633 ◽  
Author(s):  
J. Jantti ◽  
S. Keranen ◽  
J. Toikkanen ◽  
E. Kuismanen ◽  
C. Ehnholm ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Mammalian Cells ◽  
Intracellular Transport ◽  
Secretory Pathway ◽  
Semliki Forest Virus ◽  
Signal Sequence ◽  
Insertion Site ◽  
Membrane Insertion ◽  
Membrane Anchor

Proteins of the syntaxin family are suggested to play a key role in determining the specificity of intracellular membrane fusion events. They belong to the class of membrane proteins which are devoid of N-terminal signal sequence and have a C-terminal membrane anchor. Sso2p is a syntaxin homologue involved in the Golgi to plasma membrane vesicular transport in yeast. The protein was transiently expressed in BHK-21 cells using the Semliki Forest virus vector, and its localization and mode of membrane insertion were studied. By immunofluorescence and immuno-EM we show that Sso2p is transported to its final location, the plasma membrane, along the biosynthetic pathway. Experiments with synchronized Sso2p synthesis or expression of the protein in the presence of brefeldin A indicate endoplasmic reticulum as the initial membrane insertion site. During a 20 degrees C temperature block Sso2p accumulated in the Golgi complex and was chased to the plasma membrane by a subsequent 37 degrees C incubation in the presence of cycloheximide. The in vitro translated protein was able to associate with dog pancreatic microsomes post-translationally. A truncated form of Sso2p lacking the putative membrane anchor was used to show that this sequence is necessary for the membrane insertion in vivo and in vitro. The results show that this syntaxin-like protein does not directly associate with its target membrane but uses the secretory pathway to reach its cellular location, raising interesting questions concerning regulation of SNARE-type protein function.

scholarly journals Erg30, a Vap-33–Related Protein, Functions in Protein Transport Mediated by Copi Vesicles

1999 ◽  
Vol 146 (2) ◽  
pp. 301-312 ◽  
Author(s):  
Lior Soussan ◽  
Darya Burakov ◽  
Mathew P. Daniels ◽  
Mira Toister-Achituv ◽  
Amir Porat ◽  
...  
Keyword(s):  
Intracellular Transport ◽  
Secretory Pathway ◽  
Protein Transport ◽  
Structural Characteristics ◽  
Coiled Coil ◽  
Integral Membrane Protein ◽  
Type Ii ◽  
Golgi Transport ◽  
Protein Functions

Intracellular transport of newly synthesized and mature proteins via vesicles is controlled by a large group of proteins. Here we describe a ubiquitous rat protein—endoplasmic reticulum (ER) and Golgi 30-kD protein (ERG30)—which shares structural characteristics with VAP-33, a 33-kD protein from Aplysia californica which was shown to interact with the synaptic protein VAMP. The transmembrane topology of the 30-kD ERG30 corresponds to a type II integral membrane protein, whose cytoplasmic NH2 terminus contains a predicted coiled-coil motif. We localized ERG30 to the ER and to pre-Golgi intermediates by biochemical and immunocytochemical methods. Consistent with a role in vesicular transport, anti-ERG30 antibodies specifically inhibit intra-Golgi transport in vitro, leading to significant accumulation of COPI-coated vesicles. It appears that ERG30 functions early in the secretory pathway, probably within the Golgi and between the Golgi and the ER.

scholarly journals Lysosome biogenesis requires Rab9 function and receptor recycling from endosomes to the trans-Golgi network.

1994 ◽  
Vol 125 (3) ◽  
pp. 573-582 ◽  
Author(s):  
M A Riederer ◽  
T Soldati ◽  
A D Shapiro ◽  
J Lin ◽  
S R Pfeffer
Keyword(s):  
Lysosomal Enzyme ◽  
Lysosomal Enzymes ◽  
Secretory Pathway ◽  
Protein Transport ◽  
Fluid Phase ◽  
Dominant Negative ◽  
Lysosome Biogenesis ◽  
Dominant Negative Form ◽  
Golgi Network

Newly synthesized lysosomal enzymes bind to mannose 6-phosphate receptors (MPRs) in the TGN, and are carried to prelysosomes, where they are released. MPRs then return to the TGN for another round of transport. Rab9 is a ras-like GTPase which facilitates MPR recycling to the TGN in vitro. We show here that a dominant negative form of rab9, rab9 S21N, strongly inhibited MPR recycling in living cells. The block was specific in that the rates of biosynthetic protein transport, fluid phase endocytosis and receptor-mediated endocytosis were unchanged. Expression of rab9 S21N was accompanied by a decrease in the efficiency of lysosomal enzyme sorting. Cells compensated for the presence of the mutant protein by inducing the synthesis of both soluble and membrane-associated lysosomal enzymes, and by internalizing lysosomal enzymes that were secreted by default. These data show that MPRs are limiting in the secretory pathway of cells expressing rab9 S21N and document the importance of MPR recycling and the rab9 GTPase for efficient lysosomal enzyme delivery.

scholarly journals α-Synuclein and ALPS motifs are membrane curvature sensors whose contrasting chemistry mediates selective vesicle binding

2011 ◽  
Vol 194 (1) ◽  
pp. 89-103 ◽  
Author(s):  
Iwona M. Pranke ◽  
Vincent Morello ◽  
Joëlle Bigay ◽  
Kimberley Gibson ◽  
Jean-Marc Verbavatz ◽  
...  
Keyword(s):  
Secretory Pathway ◽  
Direct Interaction ◽  
Membrane Curvature ◽  
Yeast Cells ◽  
Amphipathic Helices ◽  
Early Secretory Pathway ◽  
Intrinsic Capacity ◽  
In Cells

Membrane curvature sensors have diverse structures and chemistries, suggesting that they might have the intrinsic capacity to discriminate between different types of vesicles in cells. In this paper, we compare the in vitro and in vivo membrane-binding properties of two curvature sensors that form very different amphipathic helices: the amphipathic lipid-packing sensor (ALPS) motif of a Golgi vesicle tether and the synaptic vesicle protein α-synuclein, a causative agent of Parkinson’s disease. We demonstrate the mechanism by which α-synuclein senses membrane curvature. Unlike ALPS motifs, α-synuclein has a poorly developed hydrophobic face, and this feature explains its dual sensitivity to negatively charged lipids and to membrane curvature. When expressed in yeast cells, these two curvature sensors were targeted to different classes of vesicles, those of the early secretory pathway for ALPS motifs and to negatively charged endocytic/post-Golgi vesicles in the case of α-synuclein. Through structures with complementary chemistries, α-synuclein and ALPS motifs target distinct vesicles in cells by direct interaction with different lipid environments.

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