scholarly journals Structure and Distribution of Organelles and Cellular Location of Calcium Transporters in Neurospora crassa

2009 ◽  
Vol 8 (12) ◽  
pp. 1845-1855 ◽  
Author(s):  
Barry J. Bowman ◽  
Marija Draskovic ◽  
Michael Freitag ◽  
Emma Jean Bowman
Keyword(s):  
Endoplasmic Reticulum ◽  
Plasma Membrane ◽  
Neurospora Crassa ◽  
Fluorescent Protein ◽  
Protein A ◽  
Vacuolar Membrane ◽  
Red Fluorescent Protein ◽  
Animal Cells ◽  
Marker Proteins ◽  
Hyphal Tip

ABSTRACT We wanted to examine the cellular locations of four Neurospora crassa proteins that transport calcium. However, the structure and distribution of organelles in live hyphae of N. crassa have not been comprehensively described. Therefore, we made recombinant genes that generate translational fusions of putative organellar marker proteins with green or red fluorescent protein. We observed putative endoplasmic reticulum proteins, encoded by grp-78 and dpm, in the nuclear envelope and associated membranes. Proteins of the vacuolar membrane, encoded by vam-3 and vma-1, were in an interconnected network of small tubules and vesicles near the hyphal tip, while in more distal regions they were in large and small spherical vacuoles. Mitochondria, visualized with tagged ARG-4, were abundant in all regions of the hyphae. Similarly, we tagged the four N. crassa proteins that transport calcium with green or red fluorescent protein to examine their cellular locations. NCA-1 protein, a homolog of the SERCA-type Ca2+-ATPase of animal cells, colocalized with the endoplasmic reticulum markers. The NCA-2 and NCA-3 proteins are homologs of Ca2+-ATPases in the vacuolar membrane in yeast or in the plasma membrane in animal cells. They colocalized with markers in the vacuolar membrane, and they also occurred in the plasma membrane in regions of the hyphae more than 1 mm from the tip. The cax gene encodes a Ca2+/H+ exchange protein found in vacuoles. As expected, the CAX protein localized to the vacuolar compartment. We observed, approximately 50 to 100 μm from the tip, a few spherical organelles that had high amounts of tagged CAX protein and tagged subunits of the vacuolar ATPase (VMA-1 and VMA-5). We suggest that this organelle, not described previously in N. crassa, may have a role in sequestering calcium.

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

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.

scholarly journals mCLCA4 ER processing and secretion requires luminal sorting motifs

2008 ◽  
Vol 295 (1) ◽  
pp. C279-C287 ◽  
Author(s):  
Chunlei Huan ◽  
Kai Su Greene ◽  
Bo Shui ◽  
Gwendolyn Spizz ◽  
Haitao Sun ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Plasma Membrane ◽  
Green Fluorescent Protein ◽  
Enhanced Green Fluorescent Protein ◽  
Fluorescent Protein ◽  
Chloride Transport ◽  
Proteolytic Processing ◽  
Regulatory Sequences ◽  
Cellular Processing ◽  
Green Fluorescent

Ca+-activated Cl− channel (CLCA) proteins are encoded by a family of highly related and clustered genes in mammals that are markedly upregulated in inflammation and have been shown to affect chloride transport. Here we describe the cellular processing and regulatory sequences underlying murine (m) CLCA4 proteins. The 125-kDa mCLCA4 gene product is cleaved to 90- and 40-kDa fragments, and the NH2- and COOH-terminal fragments are secreted, where they are found in cell media and associated with the plasma membrane. The 125-kDa full-length protein is only found in the endoplasmic reticulum (ER), and specific luminal diarginine retention and dileucine forward trafficking signals contained within the CLCA4 sequence regulate export from the ER and proteolytic processing. Mutation of the dileucine luminal sequences resulted in ER trapping of the immaturely glycosylated 125-kDa peptide, indicating that proteolytic cleavage occurs following recognition of the trafficking motifs. Moreover, the mutated dileucine and diarginine signal sequences directed processing of a secreted form of enhanced green fluorescent protein in a manner consistent with the effects on mCLCA4.

scholarly journals Sarco/endoplasmic-reticulum calcium ATPase SERCA1 is maintained in the endoplasmic reticulum by a retrieval signal located between residues 1 and 211

2003 ◽  
Vol 371 (3) ◽  
pp. 775-782 ◽  
Author(s):  
Thomas NEWTON ◽  
John P. J. BLACK ◽  
John BUTLER ◽  
Anthony G. LEE ◽  
John CHAD ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Plasma Membrane ◽  
Fluorescent Protein ◽  
Subcellular Location ◽  
Terminal Segment ◽  
Calcium Atpase ◽  
Endoplasmic Reticulum Calcium ◽  
Calcium Pumps ◽  
Green Fluorescent ◽  
Fast Twitch

The location of sarco/endoplasmic-reticulum calcium ATPase (SERCA) retention/retrieval motifs in the sequence of the SERCA1 has been investigated by examining the subcellular location in COS-7 cells of enhanced-green-fluorescent-protein-tagged calcium-pump chimaeras. These chimaeras have been constructed from the fast-twitch SERCA1 and the plasma-membrane calcium ATPase PMCA3. The N-terminal, central and C-terminal segments of these calcium pumps were exchanged between SERCA1 and PMCA3. The segments exchanged correspond to residues 1–211, 212–711 and 712–994 of SERCA1, and residues 1–264, 265–788 and 789–1159 of PMCA3 respectively. Only chimaeras containing the N-terminal segment of SERCA1 were located in the endoplasmic reticulum (ER), whereas chimaeras containing the N-terminal segment from PMCA3 were able to escape from the ER and enter the endomembrane pathway en route for the plasma membrane. Co-localization of SERCA1 in COS-7 cells with the ER/Golgi-intermediate compartment marker ERGIC53 indicates that SERCA1 is maintained in the ER by a process of retrieval. These results indicate that the N-terminal region of SERCA1, containing transmembrane helices M1 and M2, contains an ER-retrieval signal.

Analysis of Pelagia noctiluca proteome Reveals a Red Fluorescent Protein, a Zinc Metalloproteinase and a Peroxiredoxin

2017 ◽  
Vol 36 (2) ◽  
pp. 77-97 ◽  
Author(s):  
Bárbara Frazão ◽  
Alexandre Campos ◽  
Hugo Osório ◽  
Benjamin Thomas ◽  
Sérgio Leandro ◽  
...  
Keyword(s):  
Fluorescent Protein ◽  
Protein A ◽  
Red Fluorescent Protein ◽  
Pelagia Noctiluca

scholarly journals Physical and functional interaction of FgFtr1–FgFet1 and FgFtr2–FgFet2 is required for iron uptake in Fusarium graminearum

2007 ◽  
Vol 408 (1) ◽  
pp. 97-104 ◽  
Author(s):  
Yong-Sung Park ◽  
Ji-Hyun Kim ◽  
Jin-Hwa Cho ◽  
Hyo-Ihl Chang ◽  
Seung-Wook Kim ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Fusarium Graminearum ◽  
Iron Uptake ◽  
Fluorescent Protein ◽  
Amino Acid Level ◽  
Normal Function ◽  
Vacuolar Membrane ◽  
Uptake System ◽  
Genes Encoding ◽  
Green Fluorescent

FgFtr1 and FgFtr2 are putative iron permeases, and FgFet1 and FgFet2 are putative ferroxidases of Fusarium graminearum. They have high homologies with iron permease ScFtr1 and ferroxidase ScFet3 of Saccharomyces cerevisiae at the amino acid level. The genes encoding iron permease and ferroxidase were localized to the same chromosome in the manner of FgFtr1/FgFet1 and FgFtr2/FgFet2. The GFP (green fluorescent protein)-fused versions of FgFtr1 and FgFtr2 showed normal functions when compared with FgFtr1 and FgFtr2 in an S. cerevisiae system, and the cellular localizations of FgFtr1 and FgFtr2 in S. cerevisiae depended on the expression of their putative ferroxidase partners FgFet1 and FgFet2 respectively. Although FgFtr1 was found on the plasma membrane when FgFet1 and FgFtr1 were co-transformed in S. cerevisiae, most of the FgFtr1 was found in the endoplasmic reticulum compartment when co-expressed with FgFet2. Furthermore, FgFtr2 was found on the vacuolar membrane when FgFet2 was co-expressed. From the two-hybrid analysis, we confirmed the interaction of FgFtr1 and FgFet1, and the same result was found between FgFtr2 and FgFet2. Iron-uptake activity also depended on the existence of the respective partner. Finally, the FgFtr1 and FgFtr2 were found on the plasma and vacuolar membrane respectively, in F. graminearum. Taken together, these results strongly suggest that FgFtr1 and FgFtr2 from F. graminearum encode the iron permeases of the plasma membrane and vacuolar membrane respectively, and require their specific ferroxidases to carry out normal function. Furthermore, the present study suggests that the reductive iron-uptake system is conserved from yeast to filamentous fungi.

Cortical endoplasmic reticulum in plants

1990 ◽  
Vol 96 (3) ◽  
pp. 355-373
Author(s):  
P. K. HEPLER ◽  
B. A. PALEVITZ ◽  
S. A. LANCELLE ◽  
M. M. MCCAULEY ◽  
L. LICHTSCHIDL
Keyword(s):  
Endoplasmic Reticulum ◽  
Plasma Membrane ◽  
Cortical Network ◽  
Specific Function ◽  
Animal Cells ◽  
Daughter Cells ◽  
Polygonal Pattern ◽  
Membrane Tubules ◽  
The Individual ◽  
Cortical Endoplasmic Reticulum

Structural observations provide persuasive evidence for the existence of a cortical network of endoplasmic reticulum (ER) in a large number of plant and animal cells. The network in plants generally possesses a polygonal pattern in which smooth, tubular elements are joined by intervening lamellar segments. The individual elements of ER are often positioned extremely close to the plasma membrane (PM), and may form appositional contacts, but fusion does not occur. The network arises at cytokinesis and establishes continuity between the cortical ER of daughter cells in the form of tightly furled membrane tubules that traverse the plasmodesmata. The specific function of the cortical ER complex is unknown but different possibilities seem attractive. It may serve key roles in anchoring the cytoskeleton and in facilitating secretion. The cortical ER might also participate in the communication of signals between the exterior of the cell and cytoplasm. As a consequence of its ability to release and/or sequester Ca, the ER could control the cytoplasmic activity of this ion and thus a host of physiologically and developmentally important reactions.

scholarly journals The Plasma Membrane Proton Pump PMA-1 Is Incorporated into Distal Parts of the Hyphae Independently of the Spitzenkörper in Neurospora crassa

2013 ◽  
Vol 12 (8) ◽  
pp. 1097-1105 ◽  
Author(s):  
Rosa A. Fajardo-Somera ◽  
Barry Bowman ◽  
Meritxell Riquelme
Keyword(s):  
Plasma Membrane ◽  
Neurospora Crassa ◽  
Nutrient Uptake ◽  
Laser Scanning ◽  
Germ Tube ◽  
Secretory Pathway ◽  
Fluorescent Protein ◽  
Fungal Growth ◽  
Electrochemical Gradient ◽  
Content Type

ABSTRACT Most models for fungal growth have proposed a directional traffic of secretory vesicles to the hyphal apex, where they temporarily aggregate at the Spitzenkörper before they fuse with the plasma membrane (PM). The PM H + -translocating ATPase (PMA-1) is delivered via the classical secretory pathway (endoplasmic reticulum [ER] to Golgi) to the cell surface, where it pumps H + out of the cell, generating a large electrochemical gradient that supplies energy to H + -coupled nutrient uptake systems. To characterize the traffic and delivery of PMA-1 during hyphal elongation, we have analyzed by laser scanning confocal microscopy (LSCM) strains of Neurospora crassa expressing green fluorescent protein (GFP)-tagged versions of the protein. In conidia, PMA-1-GFP was evenly distributed at the PM. During germination and germ tube elongation, PMA-1-GFP was found all around the conidial PM and extended to the germ tube PM, but fluorescence was less intense or almost absent at the tip. Together, the data indicate that the electrochemical gradient driving apical nutrient uptake is generated from early developmental stages. In mature hyphae, PMA-1-GFP localized at the PM at distal regions (>120 μm) and in completely developed septa, but not at the tip, indicative of a distinct secretory route independent of the Spitzenkörper occurring behind the apex.

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