Plasma membrane phospholipid signature recruits the plant exocyst complex via the EXO70A1 subunit

Polarized exocytosis is essential for many vital processes in eukaryotic cells, where secretory vesicles are targeted to distinct plasma membrane domains characterized by their specific lipid–protein composition. Heterooctameric protein complex exocyst facilitates the vesicle tethering to a target membrane and is a principal cell polarity regulator in eukaryotes. The architecture and molecular details of plant exocyst and its membrane recruitment have remained elusive. Here, we show that the plant exocyst consists of two modules formed by SEC3–SEC5–SEC6–SEC8 and SEC10–SEC15–EXO70–EXO84 subunits, respectively, documenting the evolutionarily conserved architecture within eukaryotes. In contrast to yeast and mammals, the two modules are linked by a plant-specific SEC3–EXO70 interaction, and plant EXO70 functionally dominates over SEC3 in the exocyst recruitment to the plasma membrane. Using an interdisciplinary approach, we found that the C-terminal part of EXO70A1, the canonical EXO70 isoform in Arabidopsis, is critical for this process. In contrast to yeast and animal cells, the EXO70A1 interaction with the plasma membrane is mediated by multiple anionic phospholipids uniquely contributing to the plant plasma membrane identity. We identified several evolutionary conserved EXO70 lysine residues and experimentally proved their importance for the EXO70A1–phospholipid interactions. Collectively, our work has uncovered plant-specific features of the exocyst complex and emphasized the importance of the specific protein–lipid code for the recruitment of peripheral membrane proteins.

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CSI1, PATROL1, and exocyst complex cooperate in delivery of cellulose synthase complexes to the plasma membrane

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1800182115 ◽

2018 ◽

Vol 115 (15) ◽

pp. E3578-E3587 ◽

Cited By ~ 32

Author(s):

Xiaoyu Zhu ◽

Shundai Li ◽

Songqin Pan ◽

Xiaoran Xin ◽

Ying Gu

Keyword(s):

Plasma Membrane ◽

Live Cell Imaging ◽

Cell Imaging ◽

De Novo ◽

Cellulose Synthase ◽

Specific Protein ◽

Cellulose Synthesis ◽

Docking Site ◽

Multiple Components ◽

Exocyst Complex

Cellulose synthesis occurs exclusively at the plasma membrane by cellulose synthase complexes (CSCs). Therefore, delivery of CSCs to discrete sites at the plasma membrane is critical for cellulose synthesis. Despite their significance, the delivery of CSCs is poorly understood. Here we used proteomics approaches, functional genetics, and live cell imaging to show that the de novo secretion of CSCs is mediated by cooperation among cellulose synthase interactive 1 (CSI1), the plant-specific protein PATROL1, and exocyst complex in Arabidopsis thaliana. We propose that CSI1 plays a role in marking the docking site, which allows CSCs-containing vesicles access to the plasma membrane through its interaction with microtubules. PATROL1 assists in exocytosis by its interaction with multiple components, including CSI1, CSCs, and exocyst subunits. Both PATROL1 and the exocyst complex determine the rate of delivery of CSCs to the plasma membrane. By monitoring the exocyst complex, PATROL1, CSI1, and CSCs dynamics in real time, we present a timeline of events for exocytosis of CSCs. Our findings provide unique insights into the evolution of exocytosis in eukaryotes.

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Localization of Adenosine Triphosphatase Activity in the Phleom of Nicotiana Tabacum

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100094401 ◽

1972 ◽

Vol 30 ◽

pp. 230-231

Author(s):

James Cronshaw ◽

Jamison E. Gilder

Keyword(s):

Plasma Membrane ◽

Atpase Activity ◽

Adenosine Triphosphatase ◽

Higher Plants ◽

High Surface ◽

Root Tip ◽

Animal Cells ◽

Physiological Processes ◽

Tip Cells ◽

Atpase Localization

Adenosine triphosphatase (ATPase) activity has been shown to be associated with numerous physiological processes in both plants and animal cells. Biochemical studies have shown that in higher plants ATPase activity is high in cell wall preparations and is associated with the plasma membrane, nuclei, mitochondria, chloroplasts and lysosomes. However, there have been only a few ATPase localization studies of higher plants at the electron microscope level. Poux (1967) demonstrated ATPase activity associated with most cellular organelles in the protoderm cells of Cucumis roots. Hall (1971) has demonstrated ATPase activity in root tip cells of Zea mays. There was high surface activity largely associated with the plasma membrane and plasmodesmata. ATPase activity was also demonstrated in mitochondria, dictyosomes, endoplasmic reticulum and plastids.

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Immunocytochemical localization of p65 with one-nanometer gold particles following silver development

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s042482010015719x ◽

1989 ◽

Vol 47 ◽

pp. 1042-1043

Author(s):

Richard W. Burry ◽

Diane M. Hayes

Keyword(s):

Plasma Membrane ◽

Bovine Serum Albumin ◽

Calf Serum ◽

Specific Protein ◽

Immunocytochemical Localization ◽

Electron Microscopic ◽

Gold Particles ◽

Pass Through ◽

Label Distribution ◽

Phosphate Buffered Saline

Electron microscopic (EM) immunocytochemistry localization of the neuron specific protein p65 could show which organelles contain this antigen. Antibodies (Ab) labeled with horseradish peroxidase (HRP) followed by chromogen development show a broad diffuse label distribution within cells and restricting identification of organelles. Particulate label (e.g. 10 nm colloidal gold) is highly desirable but not practical because penetration into cells requires destroying the plasma membrane. We report pre-embedding immunocytochemistry with a particulate marker, 1 nm gold, that will pass through membranes treated with saponin, a mild detergent.Cell cultures of the rat cerebellum were fixed in buffered 4% paraformaldehyde and 0.1% glutaraldehyde (Glut.). The buffer for all incubations and rinses was phosphate buffered saline with: 1% calf serum, 0.2% saponin, 0.1% gelatin, 50 mM glycine 1 mg/ml bovine serum albumin, and (not in the HRP labeled cultures) 0.02% sodium azide. The monoclonal #48 to p65 was used with three label systems: HRP, 1 nm avidin gold with IntenSE M development, and 1 nm avidin gold with Danscher development.

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Faculty Opinions recommendation of Raft nanodomains contribute to Akt/PKB plasma membrane recruitment and activation.

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

10.3410/f.1120447.576644 ◽

2009 ◽

Author(s):

Kees Jalink

Keyword(s):

Plasma Membrane ◽

Membrane Recruitment

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Increasing kinase domain proximity promotes MST2 autophosphorylation during Hippo signaling

Journal of Biological Chemistry ◽

10.1074/jbc.ra120.015723 ◽

2020 ◽

Vol 295 (47) ◽

pp. 16166-16179

Author(s):

Thao Tran ◽

Jaba Mitra ◽

Taekjip Ha ◽

Jennifer M. Kavran

Keyword(s):

Single Molecule ◽

Hippo Pathway ◽

Signal Propagation ◽

Kinase Domain ◽

Specific Protein ◽

Hippo Signaling ◽

Membrane Recruitment ◽

Chemically Induced Dimerization ◽

The Hippo Pathway

The Hippo pathway plays an important role in developmental biology, mediating organ size by controlling cell proliferation through the activity of a core kinase cassette. Multiple upstream events activate the pathway, but how each controls this core kinase cassette is not fully understood. Activation of the core kinase cassette begins with phosphorylation of the kinase MST1/2 (also known as STK3/4). Here, using a combination of in vitro biochemistry and cell-based assays, including chemically induced dimerization and single-molecule pulldown, we revealed that increasing the proximity of adjacent kinase domains, rather than formation of a specific protein assembly, is sufficient to trigger autophosphorylation. We validate this mechanism in cells and demonstrate that multiple events associated with the active pathway, including SARAH domain–mediated homodimerization, membrane recruitment, and complex formation with the effector protein SAV1, each increase the kinase domain proximity and autophosphorylation of MST2. Together, our results reveal that multiple and distinct upstream signals each utilize the same common molecular mechanism to stimulate MST2 autophosphorylation. This mechanism is likely conserved among MST2 homologs. Our work also highlights potential differences in Hippo signal propagation between each activating event owing to differences in the dynamics and regulation of each protein ensemble that triggers MST2 autophosphorylation and possible redundancy in activation.

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Diacylglycerol activates the influx of extracellular cations in T-lymphocytes independently of intracellular calcium-store depletion and possibly involving endogenous TRP6 gene products

Biochemical Journal ◽

10.1042/bj3640245 ◽

2002 ◽

Vol 364 (1) ◽

pp. 245-254 ◽

Cited By ~ 60

Author(s):

Alessandra GAMBERUCCI ◽

Emanuele GIURISATO ◽

Paola PIZZO ◽

Maristella TASSI ◽

Roberta GIUNTI ◽

...

Keyword(s):

Plasma Membrane ◽

T Lymphocytes ◽

Peripheral Blood ◽

Transient Receptor Potential ◽

Human Peripheral Blood ◽

Receptor Potential ◽

Animal Cells ◽

Bivalent Cation ◽

Intracellular Calcium Store ◽

Transient Receptor

In Jurkat and human peripheral blood T-lymphocytes, 1-oleoyl-2-acetyl-sn-glycerol (OAG), a membrane-permeant analogue of diacylglycerol, activated the influx of Ca2+, Ba2+ and Sr2+. OAG also caused plasma-membrane depolarization in Ca2+-free media that was recovered by the addition of bivalent cation, indicating the activation of Na+ influx. OAG-induced cation influx was (i) mimicked by the natural dacylglycerol 1-stearoyl-2-arachidonyl-sn-glycerol, (ii) not blocked by inhibiting protein kinase C or in the absence of phopholipase C activity and (iii) blocked by La3+ and Gd3+. Differently from OAG, both thapsigargin and phytohaemagglutinin activated a potent influx of Ca2+, but little influx of Ba2+ and Sr2+. Moreover, the influx of Ca2+ activated by thapsigargin and that activated by OAG were additive. Furthermore, several drugs (i.e. econazole, SKF96365, carbonyl cyanide p-trifluoromethoxyphenylhydrazone, 2-aminoethoxy diphenylborate and calyculin-A), while inhibiting the influx of Ca2+ induced by both thapsigargin and phytohaemagglutinin, did not affect OAG-stimulated cation influx. Transient receptor potential (TRP) 3 and TRP6 proteins have been shown previously to be activated by diacylglycerol when expressed heterologously in animal cells [Hofmann, Obukhov, Schaefer, Harteneck, Gudermann and Schultz (1999) Nature (London) 397, 259–263]. In both Jurkat and peripheral blood T-lymphocytes, mRNA encoding TRP proteins 1, 3, 4 and 6 was detected by reverse transcriptase PCR, and the TRP6 protein was detected by Western blotting in a purified plasma-membrane fraction. We conclude that T-cells express a diacylglycerol-activated cation channel, unrelated to the channel involved in capacitative Ca2+ entry, and associated with the expression of TRP6 protein.

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Visualization of the exocyst complex dynamics at the plasma membrane of Arabidopsis thaliana

Molecular Biology of the Cell ◽

10.1091/mbc.e12-06-0492 ◽

2013 ◽

Vol 24 (4) ◽

pp. 510-520 ◽

Cited By ~ 64

Author(s):

Matyáš Fendrych ◽

Lukáš Synek ◽

Tamara Pečenková ◽

Edita Janková Drdová ◽

Juraj Sekereš ◽

...

Keyword(s):

Plasma Membrane ◽

Fluorescent Protein ◽

Complex Dynamics ◽

Active Regions ◽

Snare Complex ◽

Epifluorescence Microscopy ◽

Rab Gtpases ◽

Exocyst Complex ◽

Protein Receptor ◽

Outer Domain

The exocyst complex, an effector of Rho and Rab GTPases, is believed to function as an exocytotic vesicle tether at the plasma membrane before soluble N-ethylmaleimide–sensitive factor attachment protein receptor (SNARE) complex formation. Exocyst subunits localize to secretory-active regions of the plasma membrane, exemplified by the outer domain of Arabidopsis root epidermal cells. Using variable-angle epifluorescence microscopy, we visualized the dynamics of exocyst subunits at this domain. The subunits colocalized in defined foci at the plasma membrane, distinct from endocytic sites. Exocyst foci were independent of cytoskeleton, although prolonged actin disruption led to changes in exocyst localization. Exocyst foci partially overlapped with vesicles visualized by VAMP721 v-SNARE, but the majority of the foci represent sites without vesicles, as indicated by electron microscopy and drug treatments, supporting the concept of the exocyst functioning as a dynamic particle. We observed a decrease of SEC6–green fluorescent protein foci in an exo70A1 exocyst mutant. Finally, we documented decreased VAMP721 trafficking to the plasma membrane in exo70A1 and exo84b mutants. Our data support the concept that the exocyst-complex subunits dynamically dock and undock at the plasma membrane to create sites primed for vesicle tethering.

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