Regulated Degradation of an Endoplasmic Reticulum Membrane Protein in a Tubular Lysosome in Leishmania mexicana

The cell surface of the human parasite Leishmania mexicana is coated with glycosylphosphatidylinositol (GPI)-anchored macromolecules and free GPI glycolipids. We have investigated the intracellular trafficking of green fluorescent protein- and hemagglutinin-tagged forms of dolichol-phosphate-mannose synthase (DPMS), a key enzyme in GPI biosynthesis in L. mexicana promastigotes. These functionally active chimeras are found in the same subcompartment of the endoplasmic reticulum (ER) as endogenous DPMS but are degraded as logarithmically growing promastigotes reach stationary phase, coincident with the down-regulation of endogenous DPMS activity and GPI biosynthesis in these cells. We provide evidence that these chimeras are constitutively transported to and degraded in a novel multivesicular tubule (MVT) lysosome. This organelle is a terminal lysosome, which is labeled with the endocytic marker FM 4-64, contains lysosomal cysteine and serine proteases and is disrupted by lysomorphotropic agents. Electron microscopy and subcellular fractionation studies suggest that the DPMS chimeras are transported from the ER to the lumen of the MVT via the Golgi apparatus and a population of 200-nm multivesicular bodies. In contrast, soluble ER proteins are not detectably transported to the MVT lysosome in either log or stationary phase promastigotes. Finally, the increased degradation of the DPMS chimeras in stationary phase promastigotes coincides with an increase in the lytic capacity of the MVT lysosome and changes in the morphology of this organelle. We conclude that lysosomal degradation of DPMS may be important in regulating the cellular levels of this enzyme and the stage-dependent biosynthesis of the major surface glycolipids of these parasites.

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Mitochondrial localization of Dictyostelium discoideum dUTPase mediated by its N-terminus

BMC Research Notes ◽

10.1186/s13104-019-4879-7 ◽

2020 ◽

Vol 13 (1) ◽

Author(s):

Catherine P. Chia ◽

Noriko Inoguchi ◽

Kyle C. Varon ◽

Bradley M. Bartholomai ◽

Hideaki Moriyama

Keyword(s):

Dictyostelium Discoideum ◽

Active Sites ◽

Fluorescent Protein ◽

Subcellular Fractionation ◽

Unicellular Eukaryote ◽

Gene Encoding ◽

Conserved Motifs ◽

N Terminus ◽

Key Enzyme ◽

Green Fluorescent

Abstract Objective The nuclear and mitochondrial genomes of Dictyostelium discoideum, a unicellular eukaryote, have relatively high A+T-contents of 77.5% and 72.65%, respectively. To begin to investigate how the pyrimidine biosynthetic pathway fulfills the demand for dTTP, we determined the catalytic properties and structure of the key enzyme deoxyuridine triphosphate nucleotidohydrolase (dUTPase) that hydrolyzes dUTP to dUMP, the precursor of dTTP. Results The annotated genome of D. discoideum identifies a gene encoding a polypeptide containing the five conserved motifs of homotrimeric dUTPases. Recombinant proteins, comprised of either full-length or core polypeptides with all conserved motifs but lacking residues 1-37 of the N-terminus, were active dUTPases. Crystallographic analyses of the core enzyme indicated that the C-termini, normally flexible, were constrained by interactions with the shortened N-termini that arose from the loss of residues 1-37. This allowed greater access of dUTP to active sites, resulting in enhanced catalytic parameters. A tagged protein comprised of the N-terminal forty amino acids of dUTPase fused to green fluorescent protein (GFP) was expressed in D. discoideum cells. Supporting a prediction of mitochondrial targeting information within the N-terminus, localization and subcellular fractionation studies showed GFP to be in mitochondria. N-terminal sequencing of immunoprecipitated GFP revealed the loss of the dUTPase sequence upon import into the organelle.

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Mitochondrial localization of Dictyostelium discoideum dUTPase mediated by its N-terminus

10.21203/rs.2.13015/v2 ◽

2019 ◽

Author(s):

Catherine Chia ◽

Noriko Inoguchi ◽

Kyle C. Varon ◽

Bradley M. Bartholomai ◽

Hideaki Moriyama

Keyword(s):

Dictyostelium Discoideum ◽

Active Sites ◽

Fluorescent Protein ◽

Subcellular Fractionation ◽

Unicellular Eukaryote ◽

Gene Encoding ◽

Conserved Motifs ◽

N Terminus ◽

Key Enzyme ◽

Green Fluorescent

Abstract Objective The nuclear and mitochondrial genomes of Dictyostelium discoideum , a unicellular eukaryote, have relatively high A+T-contents of 77.5% and 72.65%, respectivey. To begin to investigate how the pyrimidine biosynthetic pathway fulfills the demand for dTTP, we determined the catalytic properties and structure of the key enzyme deoxyuridine triphosphate nucleotidohydrolase (dUTPase) that hydrolyzes dUTP to dUMP, the precursor of dTTP. Results The annotated genome of D. discoideum identifies a gene encoding a polypeptide containing the five conserved motifs of homotrimeric dUTPases. Recombinant proteins, comprised of either full-length or core polypeptides with all conserved motifs but lacking residues 1-37 of the N-terminus, were active dUTPases. Crystallographic analyses of the core enzyme indicated that the C-termini, normally flexible, were constrained by interactions with the truncated N-termini. This allowed greater access of dUTP to active sites, resulting in enhanced catalytic parameters. A tagged protein comprised of the N-terminal forty amino acids of dUTPase fused to green fluorescent protein (GFP) was expressed in D. discoideum cells. Supporting a prediction of mitochondrial targeting information within the N-terminus, localization and subcellular fractionation studies showed GFP to be in mitochondria. N-terminal sequencing of immunoprecipitated GFP revealed the loss of the dUTPase sequence upon import into the organelle.

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Mitochondrial localization of Dictyostelium discoideum dUTPase mediated by its N-terminus

10.21203/rs.2.13015/v1 ◽

2019 ◽

Author(s):

Catherine Chia ◽

Noriko Inoguchi ◽

Kyle C. Varon ◽

Bradley M. Bartholomai ◽

Hideaki Moriyama

Keyword(s):

Dictyostelium Discoideum ◽

Active Sites ◽

Fluorescent Protein ◽

Subcellular Fractionation ◽

Unicellular Eukaryote ◽

Gene Encoding ◽

Conserved Motifs ◽

N Terminus ◽

Key Enzyme ◽

Green Fluorescent

Abstract Objective The nuclear and mitochondrial genomes of Dictyostelium discoideum , a unicellular eukaryote, have relatively high A+T-contents of 77.5% and 72.65%, respectivey. To begin to investigate how the pyrimidine biosynthetic pathway fulfills the demand for dTTP, we determined the catalytic properties and structure of the key enzyme deoxyuridine triphosphate nucleotidohydrolase (dUTPase) that hydrolyzes dUTP to dUMP, the precursor of dTTP. Results The annotated genome of D. discoideum identifies a gene encoding a polypeptide containing the five conserved motifs of homotrimeric dUTPases. Recombinant proteins, comprised of either full-length or core polypeptides with all conserved motifs but lacking residues 1-37 of the N-terminus, were active dUTPases. Crystallographic analyses of the core enzyme indicated that the C-termini, normally flexible, were constrained by interactions with the truncated N-termini. This allowed greater access of dUTP to active sites, resulting in enhanced catalytic parameters. A tagged protein comprised of the N-terminal forty amino acids of dUTPase fused to green fluorescent protein (GFP) was expressed in D. discoideum cells. Supporting a prediction of mitochondrial targeting information within the N-terminus, localization and subcellular fractionation studies showed GFP to be in mitochondria. N-terminal sequencing of immunoprecipitated GFP revealed the loss of the dUTPase sequence upon import into the organelle.

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Rer1p, a Retrieval Receptor for Endoplasmic Reticulum Membrane Proteins, Is Dynamically Localized to the Golgi Apparatus by Coatomer

The Journal of Cell Biology ◽

10.1083/jcb.152.5.935 ◽

2001 ◽

Vol 152 (5) ◽

pp. 935-944 ◽

Cited By ~ 102

Author(s):

Ken Sato ◽

Miyuki Sato ◽

Akihiko Nakano

Keyword(s):

Endoplasmic Reticulum ◽

Green Fluorescent Protein ◽

Membrane Proteins ◽

Golgi Apparatus ◽

Fluorescent Protein ◽

Transmembrane Domain ◽

Endoplasmic Reticulum Membrane ◽

Golgi Membrane ◽

Green Fluorescent

Rer1p, a yeast Golgi membrane protein, is required for the retrieval of a set of endoplasmic reticulum (ER) membrane proteins. We present the first evidence that Rer1p directly interacts with the transmembrane domain (TMD) of Sec12p which contains a retrieval signal. A green fluorescent protein (GFP) fusion of Rer1p rapidly cycles between the Golgi and the ER. Either a lesion of coatomer or deletion of the COOH-terminal tail of Rer1p causes its mislocalization to the vacuole. The COOH-terminal Rer1p tail interacts in vitro with a coatomer complex containing α and γ subunits. These findings not only give the proof that Rer1p is a novel type of retrieval receptor recognizing the TMD in the Golgi but also indicate that coatomer actively regulates the function and localization of Rer1p.

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The Vaccinia Virus B5 Protein Requires A34 for Efficient Intracellular Trafficking from the Endoplasmic Reticulum to the Site of Wrapping and Incorporation into Progeny Virions

Journal of Virology ◽

10.1128/jvi.01971-07 ◽

2007 ◽

Vol 82 (5) ◽

pp. 2161-2169 ◽

Cited By ~ 26

Author(s):

Amalia K. Earley ◽

Winnie M. Chan ◽

Brian M. Ward

Keyword(s):

Endoplasmic Reticulum ◽

Vaccinia Virus ◽

Intracellular Trafficking ◽

Enhanced Green Fluorescent Protein ◽

Fluorescent Protein ◽

Subcellular Fractionation ◽

Gfp Fluorescence ◽

Virion Morphogenesis ◽

Green Fluorescent

ABSTRACT The glycoproteins encoded by the vaccinia virus A34R and B5R genes are involved in intracellular envelope virus formation and are highly conserved among orthopoxviruses. A recombinant virus that has the A34R gene deleted and the B5R gene replaced with a B5R gene fused to the enhanced green fluorescent protein (B5R-GFP) gene was created (vB5R-GFP/ΔA34R) to investigate the role of A34 during virion morphogenesis. Cells infected with vB5R-GFP/ΔA34R displayed GFP fluorescence throughout the cytoplasm, which differed markedly from that seen in cells infected with a normal B5R-GFP-expressing virus (vB5R-GFP). Immunofluorescence and subcellular fractionation demonstrated that B5-GFP localizes with the endoplasmic reticulum in the absence of A34. Expression of either full-length A34 or a construct consisting of the lumenal and transmembrane domains restored normal trafficking of B5-GFP to the site of wrapping in the juxtanuclear region. Coimmunoprecipitation studies confirmed that B5 and A34 interact through their luminal domains, and further analysis revealed that in the absence of A34, B5 is not efficiently incorporated into virions released from the cell.

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Targeting of inositol 1,4,5-trisphosphate receptor to the endoplasmic reticulum by its first transmembrane domain

Biochemical Journal ◽

10.1042/bj20091051 ◽

2009 ◽

Vol 425 (1) ◽

pp. 61-74 ◽

Cited By ~ 6

Author(s):

Evangelia Pantazaka ◽

Colin W. Taylor

Keyword(s):

Endoplasmic Reticulum ◽

Fluorescent Protein ◽

Transmembrane Domain ◽

Signal Sequence ◽

Subcellular Fractionation ◽

Hydrophobic Residues ◽

Inositol 1,4,5 Trisphosphate ◽

Stable Association ◽

Green Fluorescent ◽

Trisphosphate Receptor

Targeting of IP3R (inositol 1,4,5-trisphosphate receptors) to membranes of the ER (endoplasmic reticulum) and their retention within ER or trafficking to other membranes underlies their ability to generate spatially organized Ca2+ signals. N-terminal fragments of IP3R1 (type 1 IP3R) were tagged with enhanced green fluorescent protein, expressed in COS-7 cells and their distribution was determined by confocal microscopy and subcellular fractionation. Localization of IP3R1 in the ER requires translation of between 26 and 34 residues beyond the end of the first transmembrane domain (TMD1), a region that includes TMD2 (second transmembrane domain). Replacement of these post-TMD1 residues with unrelated sequences of similar length (24–36 residues) partially mimicked the native residues. We conclude that for IP3R approx. 30 residues after TMD1 must be translated to allow a signal sequence within TMD1 to be extruded from the ribosome and mediate co-translational targeting to the ER. Hydrophobic residues within TMD1 and TMD2 then ensure stable association with the ER membrane.

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Synaptotagmins at the endoplasmic reticulum–plasma membrane contact sites maintain diacylglycerol homeostasis during abiotic stress

The Plant Cell ◽

10.1093/plcell/koab122 ◽

2021 ◽

Author(s):

Noemi Ruiz-Lopez ◽

Jessica Pérez-Sancho ◽

Alicia Esteban del Valle ◽

Richard P Haslam ◽

Steffen Vanneste ◽

...

Keyword(s):

Endoplasmic Reticulum ◽

Plasma Membrane ◽

Abiotic Stress ◽

Fluorescent Protein ◽

Mechanical Damage ◽

Membrane Contact Sites ◽

Contact Sites ◽

Membrane Contact ◽

Green Fluorescent

Abstract Endoplasmic reticulum-plasma membrane contact sites (ER-PM CS) play fundamental roles in all eukaryotic cells. Arabidopsis thaliana mutants lacking the ER-PM protein tether synaptotagmin1 (SYT1) exhibit decreased plasma membrane (PM) integrity under multiple abiotic stresses such as freezing, high salt, osmotic stress and mechanical damage. Here, we show that, together with SYT1, the stress-induced SYT3 is an ER-PM tether that also functions in maintaining PM integrity. The ER-PM CS localization of SYT1 and SYT3 is dependent on PM phosphatidylinositol-4-phosphate and is regulated by abiotic stress. Lipidomic analysis revealed that cold stress increased the accumulation of diacylglycerol at the PM in a syt1/3 double mutant relative to wild type while the levels of most glycerolipid species remain unchanged. Additionally, the SYT1-green fluorescent protein (GFP) fusion preferentially binds diacylglycerol in vivo with little affinity for polar glycerolipids. Our work uncovers a SYT-dependent mechanism of stress adaptation counteracting the detrimental accumulation of diacylglycerol at the PM produced during episodes of abiotic stress.

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Rtn1p Is Involved in Structuring the Cortical Endoplasmic Reticulum

Molecular Biology of the Cell ◽

10.1091/mbc.e06-01-0080 ◽

2006 ◽

Vol 17 (7) ◽

pp. 3009-3020 ◽

Cited By ~ 90

Author(s):

Johan-Owen De Craene ◽

Jeff Coleman ◽

Paula Estrada de Martin ◽

Marc Pypaert ◽

Scott Anderson ◽

...

Keyword(s):

Endoplasmic Reticulum ◽

Nuclear Envelope ◽

Fluorescent Protein ◽

Cell Cortex ◽

Proteomic Screen ◽

Wide Range ◽

Membrane Spanning ◽

Green Fluorescent ◽

Hydrophilic Loop ◽

Yeast Mutants

The endoplasmic reticulum (ER) contains both cisternal and reticular elements in one contiguous structure. We identified rtn1Δ in a systematic screen for yeast mutants with altered ER morphology. The ER in rtn1Δ cells is predominantly cisternal rather than reticular, yet the net surface area of ER is not significantly changed. Rtn1-green fluorescent protein (GFP) associates with the reticular ER at the cell cortex and with the tubules that connect the cortical ER to the nuclear envelope, but not with the nuclear envelope itself. Rtn1p overexpression also results in an altered ER structure. Rtn proteins are found on the ER in a wide range of eukaryotes and are defined by two membrane-spanning domains flanking a conserved hydrophilic loop. Our results suggest that Rtn proteins may direct the formation of reticulated ER. We independently identified Rtn1p in a proteomic screen for proteins associated with the exocyst vesicle tethering complex. The conserved hydophilic loop of Rtn1p binds to the exocyst subunit Sec6p. Overexpression of this loop results in a modest accumulation of secretory vesicles, suggesting impaired exocyst function. The interaction of Rtn1p with the exocyst at the bud tip may trigger the formation of a cortical ER network in yeast buds.

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Identification of a Novel Saturable Endoplasmic Reticulum Localization Mechanism Mediated by the C-Terminus of aDictyostelium Protein Disulfide Isomerase

Molecular Biology of the Cell ◽

10.1091/mbc.11.10.3469 ◽

2000 ◽

Vol 11 (10) ◽

pp. 3469-3484 ◽

Cited By ~ 35

Author(s):

Jean Monnat ◽

Eva M. Neuhaus ◽

Marius S. Pop ◽

David M. Ferrari ◽

Barbara Kramer ◽

...

Keyword(s):

Endoplasmic Reticulum ◽

Protein Disulfide Isomerase ◽

Fluorescent Protein ◽

Null Mutation ◽

Disulfide Isomerase ◽

Isomerase Activity ◽

C Terminus ◽

Complementary Action ◽

Green Fluorescent ◽

Protein Disulfide

Localization of soluble endoplasmic reticulum (ER) resident proteins is likely achieved by the complementary action of retrieval and retention mechanisms. Whereas the machinery involving the H/KDEL and related retrieval signals in targeting escapees back to the ER is well characterized, other mechanisms including retention are still poorly understood. We have identified a protein disulfide isomerase (Dd-PDI) lacking the HDEL retrieval signal normally found at the C terminus of ER residents in Dictyostelium discoideum. Here we demonstrate that its 57 residue C-terminal domain is necessary for intracellular retention of Dd-PDI and sufficient to localize a green fluorescent protein (GFP) chimera to the ER, especially to the nuclear envelope. Dd-PDI and GFP-PDI57 are recovered in similar cation-dependent complexes. The overexpression of GFP-PDI57 leads to disruption of endogenous PDI complexes and induces the secretion of PDI, whereas overexpression of a GFP-HDEL chimera induces the secretion of endogenous calreticulin, revealing the presence of two independent and saturable mechanisms. Finally, low-level expression of Dd-PDI but not of PDI truncated of its 57 C-terminal residues complements the otherwise lethal yeast TRG1/PDI1 null mutation, demonstrating functional disulfide isomerase activity and ER localization. Altogether, these results indicate that the PDI57 peptide contains ER localization determinants recognized by a conserved machinery present in D. discoideum and Saccharomyces cerevisiae.

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