scholarly journals Control of protein trafficking by reversible masking of transport signals

2016 ◽  
Vol 27 (8) ◽  
pp. 1310-1319 ◽  
Author(s):  
Omer Abraham ◽  
Karnit Gotliv ◽  
Anna Parnis ◽  
Gaelle Boncompain ◽  
Franck Perez ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Protein Trafficking ◽  
Protein Transport ◽  
Protein Import ◽  
Binding Peptide ◽  
Protein Traffic ◽  
Alternative Approach ◽  
Targeting Signal ◽  
Regulated Trafficking ◽  
Streptavidin Binding

Systems that allow the control of protein traffic between subcellular compartments have been valuable in elucidating trafficking mechanisms. Most current approaches rely on ligand or light-controlled dimerization, which results in either retardation or enhancement of the transport of a reporter. We developed an alternative approach for trafficking regulation that we term “controlled unmasking of targeting elements” (CUTE). Regulated trafficking is achieved by reversible masking of the signal that directs the reporter to its target organelle, relying on the streptavidin–biotin system. The targeting signal is generated within or immediately after a 38–amino acid streptavidin-binding peptide (SBP) that is appended to the reporter. The binding of coexpressed streptavidin to SBP causes signal masking, whereas addition of biotin causes complex dissociation and triggers protein transport to the target organelle. We demonstrate the application of this approach to the control of nuclear and peroxisomal protein import and the generation of biotin-dependent trafficking through the endocytic and COPI systems. By simultaneous masking of COPI and endocytic signals, we were able to generate a synthetic pathway for efficient transport of a reporter from the plasma membrane to the endoplasmic reticulum.

Multiple pathways for mitochondrial protein traffic

2009 ◽  
Vol 390 (8) ◽  
Author(s):  
Toshiya Endo ◽  
Koji Yamano
Keyword(s):  
Outer Membrane ◽  
Protein Trafficking ◽  
Protein Import ◽  
Mitochondrial Protein ◽  
Intermembrane Space ◽  
Mitochondrial Protein Import ◽  
Inner Membrane ◽  
Protein Traffic

Abstract Mitochondria are two-membrane bounded organelles consisting of 1000–2000 different proteins, most of which are synthesized in the cytosol and subsequently imported into mitochondria. The imported proteins are further sorted to one of the four compartments, the outer membrane, intermembrane space, inner membrane, and matrix, mostly following one of the five major pathways. Mitochondrial protein import and sorting are mediated by the translocator complexes in the membranes and chaperones in the aqueous compartments operating along the import pathways. Here, we summarize the expanding knowledge on the roles of translocators, chaperones, and related components in the multiple pathways for mitochondrial protein trafficking.

scholarly journals Membrane translocation of lumenal domains of membrane proteins powered by downstream transmembrane sequences

2013 ◽  
Vol 24 (19) ◽  
pp. 3123-3132 ◽  
Author(s):  
Takaaki Yabuki ◽  
Fumiko Morimoto ◽  
Yuichiro Kida ◽  
Masao Sakaguchi
Keyword(s):  
Endoplasmic Reticulum ◽  
Membrane Proteins ◽  
Endoplasmic Reticulum Membrane ◽  
Type I ◽  
Surface Plasmon Resonance Analysis ◽  
Binding Peptide ◽  
Resonance Analysis ◽  
N Terminus ◽  
Signal Anchor ◽  
Streptavidin Binding

Translocation of the N-terminus of a type I signal anchor (SA-I) sequence across the endoplasmic reticulum membrane can be arrested by tagging with a streptavidin-binding peptide tag (SBP tag) and trapping by streptavidin. In the present study, we first examine the affinity required for the translocation arrest. When the SBP tag is serially truncated, the ability for arrest gradually decreases. Surface plasmon resonance analysis shows that an interaction as strong as 10−8 M or a smaller dissociation constant is required for trapping the topogenesis of a natural SA-I sequence. Such truncated tags, however, become effective by mutating the SA-I sequence, suggesting that the translocation motivation is considerably influenced by the properties of the SA-I sequence. In addition, we introduce the SBP tag into lumenal loops of a multispanning membrane protein, human erythrocyte band 3. Among the tagged loops between transmembrane 1 (TM1) and TM8, three loops are trapped by cytosolic streptavidin. These loops are followed by TM sequences possessing topogenic properties, like the SA-I sequence, and translocation of one loop is diminished by insertion of a proline into the following TM sequence. These findings suggest that the translocation of lumenal loops by SA-I–like TM sequences has a crucial role in topogenesis of multispanning membrane proteins.

scholarly journals Targeting endoplasmic reticulum protein transport: a novel strategy to kill malignant B cells and overcome fludarabine resistance in CLL

Blood ◽  
2006 ◽  
Vol 107 (1) ◽  
pp. 222-231 ◽  
Author(s):  
Jennifer S. Carew ◽  
Steffan T. Nawrocki ◽  
Yelena V. Krupnik ◽  
Kenneth Dunner ◽  
David J. McConkey ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
B Cells ◽  
Protein Trafficking ◽  
Protein Transport ◽  
Brefeldin A ◽  
Secretory Protein ◽  
Lymphocytic Leukemia ◽  
Mtdna Mutations ◽  
Golgi Protein ◽  
Induced Apoptosis

Abstract Previous studies showed that chronic lymphocytic leukemia (CLL) cells exhibit certain mitochondrial abnormalities including mtDNA mutations, increased superoxide generation, and aberrant mitochondrial biogenesis, which are associated with impaired apoptosis and reduced sensitivity to fludarabine. Here we report that CLL cells and multiple myeloma cells are highly sensitive to brefeldin A, an inhibitor of endoplasmic reticulum (ER) to Golgi protein transport currently being developed as a novel anticancer agent in a prodrug formulation. Of importance, brefeldin A effectively induced apoptosis in fludarabine-refractory CLL cells. Disruption of protein trafficking by brefeldin A caused the sequestration of the prosurvival factors APRIL and VEGF in the ER, leading to abnormal ER swelling and a decrease in VEGF secretion. Such ER stress and blockage of secretory protein traffic eventually resulted in Golgi collapse, activation of caspases, and cell death. Notably, the cellular sensitivity to this compound appeared to be independent of p53 status. Taken together, these findings suggest that malignant B cells may be highly dependent on ER-Golgi protein transport and that targeting this process may be a promising therapeutic strategy for B-cell malignancies, especially for those that respond poorly to conventional treatments.

Genetic interactions of Hrd3p and Der3p/Hrd1p with Sec61p suggest a retro-translocation complex mediating protein transport for ER degradation

1999 ◽  
Vol 112 (22) ◽  
pp. 4123-4134 ◽  
Author(s):  
R.K. Plemper ◽  
J. Bordallo ◽  
P.M. Deak ◽  
C. Taxis ◽  
R. Hitt ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Protein Transport ◽  
Protein Import ◽  
De Novo ◽  
Ubiquitin Proteasome System ◽  
Central Component ◽  
Secretory Proteins ◽  
Endoplasmic Reticulum Membrane ◽  
Yeast Saccharomyces Cerevisiae ◽  
Ubiquitin Proteasome

The endoplasmic reticulum contains a quality control system that subjects misfolded or unassembled secretory proteins to rapid degradation via the cytosolic ubiquitin proteasome system. This requires retrograde protein transport from the endoplasmic reticulum back to the cytosol. The Sec61 pore, the central component of the protein import channel into the endoplasmic reticulum, was identified as the core subunit of the retro-translocon as well. As import of mutated proteins into the endoplasmic reticulum lumen is successfully terminated, a new targeting mechanism must exist that mediates re-entering of misfolded proteins into the Sec61 pore from the lumenal side de novo. The previously identified proteins Der3p/Hrd1p and, as we show here, Hrd3p of the yeast Saccharomyces cerevisiae, are localised in the endoplasmic reticulum membrane and are essential for the degradation of several substrates of the endoplasmic reticulum degradation machinery. Based on genetic studies we demonstrate that they functionally interact with each other and with Sec61p, probably establishing the central part of the retro-translocon. In the absence of Hrd3p, the otherwise stable protein Der3p/Hrd1p becomes rapidly degraded. This depends on a functional ubiquitin proteasome system and the presence of substrate molecules of the endoplasmic reticulum degradation system. When overexpressed, Der3p/Hrd1p accelerates CPY* degradation in Delta(hrd3) cells. Our data suggest a recycling process of Der3p/Hrd1p through Hrd3p. The retro-translocon seems to be build up at least by the Sec61 pore, Der3p/Hrd1p and Hrd3p and mediates both retrograde transport and ubiquitination of substrate molecules.

scholarly journals Suppression of Coatomer Mutants by a New Protein Family with COPI and COPII Binding Motifs in Saccharomyces cerevisiae

2003 ◽  
Vol 14 (8) ◽  
pp. 3097-3113 ◽  
Author(s):  
Thomas Sandmann ◽  
Johannes M. Herrmann ◽  
Jörn Dengjel ◽  
Heinz Schwarz ◽  
Anne Spang
Keyword(s):  
Endoplasmic Reticulum ◽  
Protein Trafficking ◽  
Secretory Pathway ◽  
Protein Transport ◽  
Integral Membrane Protein ◽  
Eukaryotic Cell ◽  
Vesicle Formation ◽  
Binding Motifs ◽  
Early Secretory Pathway ◽  
Direct Binding

Protein trafficking is achieved by a bidirectional vesicle flow between the various compartments of the eukaryotic cell. COPII coated vesicles mediate anterograde protein transport from the endoplasmic reticulum to the Golgi apparatus, whereas retrograde Golgi-to-endoplasmic reticulum vesicles use the COPI coat. Inactivation of COPI vesicle formation in conditional sec21 (γ-COP) mutants rapidly blocks transport of certain proteins along the early secretory pathway. We have identified the integral membrane protein Mst27p as a strong suppressor of sec21-3 and ret1-1 mutants. A C-terminal KKXX motif of Mst27p that allows direct binding to the COPI complex is crucial for its suppression ability. Mst27p and its homolog Yar033w (Mst28p) are part of the same complex. Both proteins contain cytoplasmic exposed C termini that have the ability to interact directly with COPI and COPII coat complexes. Site-specific mutations of the COPI binding domain abolished suppression of the sec21 mutants. Our results indicate that overexpression of MST27 provides an increased number of coat binding sites on membranes of the early secretory pathway and thereby promotes vesicle formation. As a consequence, the amount of cargo that can bind COPI might be important for the regulation of the vesicle flow in the early secretory pathway.

scholarly journals Peroxisome Senescence in Human Fibroblasts

2002 ◽  
Vol 13 (12) ◽  
pp. 4243-4255 ◽  
Author(s):  
Julie E. Legakis ◽  
Jay I. Koepke ◽  
Chris Jedeszko ◽  
Ferdous Barlaskar ◽  
Laura J. Terlecky ◽  
...  
Keyword(s):  
Molecular Mechanisms ◽  
Protein Import ◽  
Human Fibroblasts ◽  
Toxic Metabolite ◽  
Age Related ◽  
Peroxisomal Targeting ◽  
Targeting Signal ◽  
Effects Of Aging ◽  
Antioxidant Enzyme Catalase ◽  
Import Receptor

The molecular mechanisms of peroxisome biogenesis have begun to emerge; in contrast, relatively little is known about how the organelle functions as cells age. In this report, we characterize age-related changes in peroxisomes of human cells. We show that aging compromises peroxisomal targeting signal 1 (PTS1) protein import, affecting in particular the critical antioxidant enzyme catalase. The number and appearance of peroxisomes are altered in these cells, and the organelles accumulate the PTS1-import receptor, Pex5p, on their membranes. Concomitantly, cells produce increasing amounts of the toxic metabolite hydrogen peroxide, and we present evidence that this increased load of reactive oxygen species may further reduce peroxisomal protein import and exacerbate the effects of aging.

scholarly journals Rosa canina L. Can Restore Endoplasmic Reticulum Alterations, Protein Trafficking and Membrane Integrity in a Dextran Sulfate Sodium-Induced Inflammatory Bowel Disease Phenotype

Nutrients ◽  
2021 ◽  
Vol 13 (2) ◽  
pp. 441
Author(s):  
Dalanda Wanes ◽  
Mohamad Toutounji ◽  
Hichem Sebai ◽  
Sandra Rizk ◽  
Hassan Y. Naim
Keyword(s):  
Inflammatory Bowel Disease ◽  
Endoplasmic Reticulum ◽  
Lipid Rafts ◽  
Protein Trafficking ◽  
Dextran Sulfate ◽  
Dextran Sulfate Sodium ◽  
Membrane Integrity ◽  
Membrane Glycoproteins ◽  
Rosa Canina ◽  
Inflammatory Bowel

Rosa canina L. is a natural polyphenol-rich medicinal plant that exhibits antioxidant and anti-inflammatory activities. Recent in vivo studies have demonstrated that a methanol extract of Rosa canina L. (RCME) has reversed an inflammatory bowel disease (IBD)-like phenotype that has been triggered by dextran sulfate sodium (DSS) in mice. In the current study, we investigated the effects of RCME on perturbations of cellular mechanisms induced by DSS-treatment of intestinal Caco-2 cells, including stress response in the endoplasmic reticulum (ER), protein trafficking and sorting as well as lipid rafts integrity and functional capacities of an intestinal enzyme. 6 days post-confluent cells were treated for 24 h with DSS (3%) or simultaneously with DSS (3%) and RCME (100 µg/mL) or exclusively with RCME (100 µg/mL) or not treated. The results obtained demonstrate the ability of RCME to counteract the substantial increase in the expression levels of several ER stress markers in DSS-treated cells. Concomitantly, the delayed trafficking of intestinal membrane glycoproteins sucrase-isomaltase (SI) and dipeptidyl peptidase 4 (DPP4) induced by DSS between the ER and the Golgi has been compromised by RCME. Furthermore, RCME restored the partially impaired polarized sorting of SI and DPP4 to the brush border membrane. An efficient sorting mechanism of SI and DPP4 is tightly associated with intact lipid rafts structures in the trans-Golgi network (TGN), which have been distorted by DSS and normalized by RCME. Finally, the enzymatic activities of SI are enhanced in the presence of RCME. Altogether, DSS treatment has triggered ER stress, impaired trafficking and function of membrane glycoproteins and distorted lipid rafts, all of which can be compromised by RCME. These findings indicate that the antioxidants in RCME act at two major sites in Caco-2 cells, the ER and the TGN and are thus capable of maintaining the membrane integrity by correcting the sorting of membrane-associated proteins.

scholarly journals Characterization of Mmp37p, aSaccharomyces cerevisiaeMitochondrial Matrix Protein with a Role in Mitochondrial Protein Import

2006 ◽  
Vol 17 (9) ◽  
pp. 4051-4062 ◽  
Author(s):  
Michelle R. Gallas ◽  
Mary K. Dienhart ◽  
Rosemary A. Stuart ◽  
Roy M. Long
Keyword(s):  
Matrix Protein ◽  
Protein Import ◽  
Mitochondrial Protein ◽  
Temperature Sensitive ◽  
Inner Membrane ◽  
Mitochondrial Inner Membrane ◽  
Close Proximity ◽  
The Matrix ◽  
Targeting Signal

Many mitochondrial proteins are encoded by nuclear genes and after translation in the cytoplasm are imported via translocases in the outer and inner membranes, the TOM and TIM complexes, respectively. Here, we report the characterization of the mitochondrial protein, Mmp37p (YGR046w) and demonstrate its involvement in the process of protein import into mitochondria. Haploid cells deleted of MMP37 are viable but display a temperature-sensitive growth phenotype and are inviable in the absence of mitochondrial DNA. Mmp37p is located in the mitochondrial matrix where it is peripherally associated with the inner membrane. We show that Mmp37p has a role in the translocation of proteins across the mitochondrial inner membrane via the TIM23-PAM complex and further demonstrate that substrates containing a tightly folded domain in close proximity to their mitochondrial targeting sequences display a particular dependency on Mmp37p for mitochondrial import. Prior unfolding of the preprotein, or extension of the region between the targeting signal and the tightly folded domain, relieves their dependency for Mmp37p. Furthermore, evidence is presented to show that Mmp37 may affect the assembly state of the TIM23 complex. On the basis of these findings, we hypothesize that the presence of Mmp37p enhances the early stages of the TIM23 matrix import pathway to ensure engagement of incoming preproteins with the mtHsp70p/PAM complex, a step that is necessary to drive the unfolding and complete translocation of the preprotein into the matrix.

scholarly journals Protein Import into Hydrogenosomes of Trichomonas vaginalis Involves both N-Terminal and Internal Targeting Signals: a Case Study of Thioredoxin Reductases

2008 ◽  
Vol 7 (10) ◽  
pp. 1750-1757 ◽  
Author(s):  
Marek Mentel ◽  
Verena Zimorski ◽  
Patrick Haferkamp ◽  
William Martin ◽  
Katrin Henze
Keyword(s):  
Trichomonas Vaginalis ◽  
Protein Import ◽  
Atp Synthesis ◽  
Antioxidant Systems ◽  
Low Oxygen ◽  
Oxygen Sensitive ◽  
Targeting Signal ◽  
Targeting Signals ◽  
Thioredoxin Reductases

ABSTRACT The parabasalian flagellate Trichomonas vaginalis harbors mitochondrion-related and H2-producing organelles of anaerobic ATP synthesis, called hydrogenosomes, which harbor oxygen-sensitive enzymes essential to its pyruvate metabolism. In the human urogenital tract, however, T. vaginalis is regularly exposed to low oxygen concentrations and therefore must possess antioxidant systems protecting the organellar environment against the detrimental effects of molecular oxygen and reactive oxygen species. We have identified two closely related hydrogenosomal thioredoxin reductases (TrxRs), the hitherto-missing component of a thioredoxin-linked hydrogenosomal antioxidant system. One of the two hydrogenosomal TrxR isoforms, TrxRh1, carried an N-terminal extension resembling known hydrogenosomal targeting signals. Expression of hemagglutinin-tagged TrxRh1 in transfected T. vaginalis cells revealed that its N-terminal extension was necessary to import the protein into the organelles. The second hydrogenosomal TrxR isoform, TrxRh2, had no N-terminal targeting signal but was nonetheless efficiently targeted to hydrogenosomes. N-terminal presequences from hydrogenosomal proteins with known processing sites, i.e., the alpha subunit of succinyl coenzyme A synthetase (SCSα) and pyruvate:ferredoxin oxidoreductase A, were investigated for their ability to direct mature TrxRh1 to hydrogenosomes. Neither presequence directed TrxRh1 to hydrogenosomes, indicating that neither extension is, by itself, sufficient for hydrogenosomal targeting. Moreover, SCSα lacking its N-terminal extension was efficiently imported into hydrogenosomes, indicating that this extension is not required for import of this major hydrogenosomal protein. The finding that some hydrogenosomal enzymes require N-terminal signals for import but that in others the N-terminal extension is not necessary for targeting indicates the presence of additional targeting signals within the mature subunits of several hydrogenosome-localized proteins.

The yeast SLY gene products, suppressors of defects in the essential GTP-binding Ypt1 protein, may act in endoplasmic reticulum-to-Golgi transport

1991 ◽  
Vol 11 (6) ◽  
pp. 2980-2993
Author(s):  
R Ossig ◽  
C Dascher ◽  
H H Trepte ◽  
H D Schmitt ◽  
D Gallwitz
Keyword(s):  
Endoplasmic Reticulum ◽  
Secretory Pathway ◽  
Protein Transport ◽  
Single Copy ◽  
Integral Membrane Protein ◽  
Carboxypeptidase Y ◽  
Null Mutant ◽  
Yeast Saccharomyces Cerevisiae ◽  
Golgi Transport ◽  
Gtp Binding

It has been shown previously that defects in the essential GTP-binding protein, Ypt1p, lead to a block in protein transport from the endoplasmic reticulum (ER) to the Golgi apparatus in the yeast Saccharomyces cerevisiae. Here we report that four newly discovered suppressors of YPT1 deletion (SLY1-20, SLY2, SLY12, and SLY41) to a varying degree restore ER-to-Golgi transport defects in cells lacking Ypt1p. These suppressors also partially complement the sec21-1 and sec22-3 mutants which lead to a defect early in the secretory pathway. Sly1p-depleted cells, as well as a conditional lethal sly2 null mutant at nonpermissive temperatures, accumulate ER membranes and core-glycosylated invertase and carboxypeptidase Y. The sly2 null mutant under restrictive conditions (37 degrees C) can be rescued by the multicopy suppressor SLY12 and the single-copy suppressor SLY1-20, indicating that these three SLY genes functionally interact. Sly2p is shown to be an integral membrane protein.

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