Subcellular localisation of an endoplasmic reticulum-plasma membrane tethering factor, SYNAPTOTAGMIN 1, is affected by fluorescent protein fusion

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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The endoplasmic reticulum-plasma membrane tethering protein TMEM24 is a regulator of cellular Ca2+ homeostasis

10.1101/2021.06.23.449694 ◽

2021 ◽

Author(s):

Beichen Xie ◽

Styliani Panagiotou ◽

Jing Cen ◽

Patrick Gilon ◽

Peter Bergsten ◽

...

Keyword(s):

Endoplasmic Reticulum ◽

Plasma Membrane ◽

Insulin Secretion ◽

Lipid Transfer Protein ◽

Underlying Mechanism ◽

Lipid Transfer ◽

Β Cells ◽

Transfer Protein ◽

Lipid Exchange ◽

Membrane Tethering

Endoplasmic reticulum (ER) - plasma membrane (PM) contacts are sites of lipid exchange and Ca2+ transport, and both lipid transport proteins and Ca2+ channels specifically accumulate at these locations. In pancreatic β-cells, both lipid- and Ca2+ signaling are essential for insulin secretion. The recently characterized lipid transfer protein TMEM24 dynamically localize to ER-PM contact sites and provide phosphatidylinositol, a precursor of PI(4)P and PI(4,5)P2, to the plasma membrane. β-cells lacking TMEM24 exhibit markedly suppressed glucose-induced Ca2+ oscillations and insulin secretion but the underlying mechanism is not known. We now show that TMEM24 only weakly interact with the PM, and dissociates in response to both diacylglycerol and nanomolar elevations of cytosolic Ca2+. Release of TMEM24 into the bulk ER membrane also enables direct interactions with mitochondria, and we report that loss of TMEM24 results in excessive accumulation of Ca2+ in both the ER and mitochondria and in impaired mitochondria function.

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Resistance and Adaptation to Quinidine in Saccharomyces cerevisiae: Role of QDR1 (YIL120w), Encoding a Plasma Membrane Transporter of the Major Facilitator Superfamily Required for Multidrug Resistance

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.45.5.1528-1534.2001 ◽

2001 ◽

Vol 45 (5) ◽

pp. 1528-1534 ◽

Cited By ~ 26

Author(s):

Patrı́cia A. Nunes ◽

Sandra Tenreiro ◽

Isabel Sá-Correia

Keyword(s):

Saccharomyces Cerevisiae ◽

Plasma Membrane ◽

Multidrug Resistance ◽

Fluorescent Protein ◽

Resistance Mechanisms ◽

Major Facilitator Superfamily ◽

Yeast Cells ◽

Detectable Effect ◽

Protein Fusion ◽

Major Facilitator

ABSTRACT As predicted based on structural considerations, we show results indicating that the member of the major facilitator superfamily encoded by Saccharomyces cerevisiae open reading frameYIL120w is a multidrug resistance determinant. Yil120wp was implicated in yeast resistance to ketoconazole and quinidine, but not to the stereoisomer quinine; the gene was thus named QDR1. Qdr1p was proved to alleviate the deleterious effects of quinidine, revealed by the loss of cell viability following sudden exposure of the unadapted yeast population to the drug, and to allow the earlier eventual resumption of exponential growth under quinidine stress. However, QDR1 gene expression had no detectable effect on the susceptibility of yeast cells previously adapted to quinidine. Fluorescence microscopy observation of the distribution of the Qdr1-green fluorescent protein fusion protein in living yeast cells indicated that Qdr1p is a plasma membrane protein. We also show experimental evidence indicating that yeast adaptation to growth with quinidine involves the induction of active expulsion of the drug from preloaded cells, despite the fact that this antiarrhythmic and antimalarial quinoline ring-containing drug is not present in the yeast natural environment. However, we were not able to prove that Qdr1p is directly implicated in this export. Results clearly suggest that there are other unidentified quinidine resistance mechanisms that can be used in the absence of QDR1.

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mCLCA4 ER processing and secretion requires luminal sorting motifs

AJP Cell Physiology ◽

10.1152/ajpcell.00060.2008 ◽

2008 ◽

Vol 295 (1) ◽

pp. C279-C287 ◽

Cited By ~ 6

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.

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

Eukaryotic Cell ◽

10.1128/ec.00174-09 ◽

2009 ◽

Vol 8 (12) ◽

pp. 1845-1855 ◽

Cited By ~ 73

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.

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Faculty Opinions recommendation of A plasma membrane-anchored fluorescent protein fusion illuminates sieve element plasma membranes in Arabidopsis and tobacco.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.1099325.555560 ◽

2008 ◽

Author(s):

John Patrick

Keyword(s):

Plasma Membrane ◽

Plasma Membranes ◽

Fluorescent Protein ◽

Sieve Element ◽

Protein Fusion

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The endoplasmic reticulum-plasma membrane tethering protein Ist2 regulates endoplasmic reticulum stress response in <italic>Candida albicans</italic>

Scientia Sinica Vitae ◽

10.1360/ssv-2019-0155 ◽

2019 ◽

Vol 49 (9) ◽

pp. 1165-1172

Author(s):

HangQi ZHU ◽

MingChun LI ◽

Li YANG ◽

LiPing PENG ◽

NaLi ZHU ◽

...

Keyword(s):

Endoplasmic Reticulum ◽

Candida Albicans ◽

Plasma Membrane ◽

Stress Response ◽

Endoplasmic Reticulum Stress ◽

Endoplasmic Reticulum Stress Response ◽

Membrane Tethering

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Endoplasmic Reticulum Dynamics, Inheritance, and Cytoskeletal Interactions in Budding Yeast

Molecular Biology of the Cell ◽

10.1091/mbc.01-04-0184 ◽

2002 ◽

Vol 13 (3) ◽

pp. 854-865 ◽

Cited By ~ 71

Author(s):

K. L. Fehrenbacher ◽

D. Davis ◽

M. Wu ◽

I. Boldogh ◽

Liza A. Pon

Keyword(s):

Endoplasmic Reticulum ◽

Fluorescent Protein ◽

Yeast Cells ◽

Cell Cortex ◽

Filamentous Actin ◽

Protein Fusion ◽

Directed Movement ◽

Apical Bud ◽

M Phase ◽

Green Fluorescent

The endoplasmic reticulum (ER) in Saccharomyces cerevisiae consists of a reticulum underlying the plasma membrane (cortical ER) and ER associated with the nuclear envelope (nuclear ER). We used a Sec63p-green fluorescent protein fusion protein to study motility events associated with inheritance of cortical ER and nuclear ER in living yeast cells. During M phase before nuclear migration, we observed thick, apparently rigid tubular extensions emanating from the nuclear ER that elongate, undergo sweeping motions along the cell cortex, and shorten. Two findings support a role for microtubules in this process. First, extension of tubular structures from the nuclear ER is inhibited by destabilization of microtubules. Second, astral microtubules, structures that undergo similar patterns of extension, cortical surveillance and retraction, colocalize with nuclear ER extensions. During S and G2 phases of the cell cycle, we observed anchorage of the cortical ER at the site of bud emergence and apical bud growth. Thin tubules of the ER that extend from the anchored cortical ER display undulating, apparently random movement and move into the bud as it grows. Finally, we found that cortical ER morphology is sensitive to a filamentous actin–destabilizing drug, latrunculin-A, and to mutations in the actin-encoding ACT1 gene. Our observations support 1) different mechanisms and cytoskeletal mediators for the inheritance of nuclear and cortical ER elements and 2) a mechanism for cortical ER inheritance that is cytoskeleton dependent but relies on anchorage, not directed movement.

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

Biochemical Journal ◽

10.1042/bj20021477 ◽

2003 ◽

Vol 371 (3) ◽

pp. 775-782 ◽

Cited By ~ 9

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.

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