scholarly journals Cytokinin fluoroprobe and receptor CRE1/AHK4 localize to both plasma membrane and endoplasmic reticulum

2019 ◽  
Author(s):  
Karolina Kubiasová ◽  
Juan Carlos Montesinos ◽  
Olga Šamajová ◽  
Jaroslav Nisler ◽  
Václav Mik ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Plasma Membrane ◽  
Secretory Pathway ◽  
Brefeldin A ◽  
Cell Plate ◽  
Root Apical Meristem ◽  
Transcriptional Responses ◽  
Lateral Organs ◽  
Gfp Reporter ◽  
New Perspective

The plant hormone cytokinin regulates various cell and developmental processes, including cell division and differentiation, embryogenesis, activity of shoot and root apical meristems, formation of shoot and root lateral organs and others 1. Cytokinins are perceived by a subfamily of sensor histidine kinases (HKs), which via a two-component phosphorelay cascade activate transcriptional responses in the nucleus. Based on the subcellular localization of cytokinin receptors in various transient expression systems, such as tobacco leaf epidermal cells, and membrane fractionation experiments of Arabidopsis and maize, the endoplasmic reticulum (ER) membrane has been proposed as a principal hormone perception site 2–4. Intriguingly, recent study of the cytokinin transporter PUP14 has pointed out that the plasma membrane (PM)-mediated signalling might play an important role in establishment of cytokinin response gradients in various plant organs 5. However, localization of cytokinin HK receptors to the PM, although initially suggested 6, remains ambiguous. Here, by monitoring subcellular localizations of the fluorescently labelled natural cytokinin probe iP-NBD 7 and the cytokinin receptor ARABIDOPSIS HISTIDINE KINASE 4 (CRE1/AHK4) fused to GFP reporter, we show that pools of the ER-located cytokinin fluoroprobes and receptors can enter the secretory pathway and reach the PM. We demonstrate that in cells of the root apical meristem, CRE1/AHK4 localizes to the PM and the cell plate of dividing meristematic cells. Brefeldin A (BFA) experiments revealed vesicular recycling of the receptor and its accumulation in BFA compartments. Our results provide a new perspective on cytokinin signalling and the possibility of multiple sites of perception at PM and ER, which may determine specific outputs of cytokinin signalling.

scholarly journals The secretory pathway of bovine platelets

Blood ◽  
1987 ◽  
Vol 69 (3) ◽  
pp. 878-885 ◽  
Author(s):  
JG White
Keyword(s):  
Plasma Membrane ◽  
Secretory Pathway ◽  
Human Platelet ◽  
Surface Membrane ◽  
Surrounding Medium ◽  
Human Platelets ◽  
Resting Cells ◽  
Granule Secretion ◽  
New Perspective ◽  
Platelet Secretion

Abstract Human platelets contain tortuous channels in their cytoplasm, the surface-connected or open canalicular system (OCS), that communicate directly with the surrounding medium through openings on the surface membrane. Some workers have suggested that the OCS serves as the egress route for products secreted during the release reaction. Others have proposed alternate secretory pathways. Since bovine platelets lack the OCS found in human cells, the present study has examined the secretory mechanism of these cells to see whether it can shed light on the mystery of human platelet secretion. Bovine platelet granules, in contrast to human granules, are located more peripherally in resting cells (often in contact with the plasma membrane), most do not move centrally following thrombin stimulation as do human platelet granules, and many fuse directly with the external plasma membrane without any intermediate channel. The lack of peripheral location of human granules, their central rather than peripheral movement during secretion, and the presence of extensive channels are all consistent with the larger importance of the secretory channel to human platelets. Thus, though studies of bovine secretion do show that platelets can secrete their granules by direct fusion of granule and surface membranes, other differences from human platelets emphasize that this pathway, although important to bovine platelet secretion, is less important in human platelets. Studies of bovine platelets also show that the OCS is more dynamic than might have been considered from human studies and can form rapidly in response to stimulation. Such newly formed channels are used as a conduit for secretion of granule contents. The finding emphasizes the importance of channels for granule secretion in platelets generally and puts a new perspective on the ability of these cells to form channels rapidly in response to stimulation.

scholarly journals Life and Death of Fungal Transporters Under the Challenge of Polarity

2020 ◽  
Author(s):  
Sofia Dimou ◽  
George Diallinas
Keyword(s):  
Endoplasmic Reticulum ◽  
Plasma Membrane ◽  
Membrane Proteins ◽  
Cell Polarity ◽  
Secretory Pathway ◽  
Fungal Growth ◽  
Present Evidence ◽  
Life And Death ◽  
Early Endosomes ◽  
Stress Signals

Eukaryotic plasma membrane (PM) transporters face critical challenges that are not widely present in prokaryotes. The two most important issues are proper subcellular traffic and targeting to the PM, and regulated endocytosis in response to physiological, developmental or stress signals. Sorting of transporters from their site of synthesis, the Endoplasmic Reticulum (ER), to the PM has been long thought, but not formally shown, to occur via the conventional Golgi-dependent vesicular secretory pathway. Endocytosis of specific eukaryotic transporters has been studied more systematically and shown to involve ubiquitination, internalization, and sorting to early endosomes, followed by turnover in the MVB/lysosomes/vacuole system. In specific cases internalized transporters have been shown to recycle back to the PM. However, the mechanisms of transporter forward trafficking and turnover have been overturned recently through systematic work in the model fungus Aspergillus nidulans. In this review we present evidence that shows that transporter traffic to the PM takes place through Golgi-bypass and transporter endocytosis operates via a mechanism that is distinct from that of recycling membrane cargoes essential for fungal growth. We discuss these findings in relation to adaptation to challenges imposed by cell polarity in fungi as well as in other eukaryotes and provide a rationale why transporters and possibly other housekeeping membrane proteins ‘avoid’ routes of polar trafficking.

scholarly journals Chicken Erythroid AE1 Anion Exchangers Associate with the Cytoskeleton During Recycling to the Golgi

1999 ◽  
Vol 10 (2) ◽  
pp. 455-469 ◽  
Author(s):  
Sourav Ghosh ◽  
Kathleen H. Cox ◽  
John V. Cox
Keyword(s):  
Plasma Membrane ◽  
Ammonium Chloride ◽  
Anion Exchanger ◽  
Secretory Pathway ◽  
Brefeldin A ◽  
Endocytic Pathway ◽  
Erythroid Cells ◽  
Anion Exchangers ◽  
Membrane Compartment ◽  
Chloride Treatment

Chicken erythroid AE1 anion exchangers receive endoglycosidase F (endo F)-sensitive sugar modifications in their initial transit through the secretory pathway. After delivery to the plasma membrane, anion exchangers are internalized and recycled to the Golgi where they acquire additional N-linked modifications that are resistant to endo F. During recycling, some of the anion exchangers become detergent insoluble. The acquisition of detergent insolubility correlates with the association of the anion exchanger with cytoskeletal ankyrin. Reagents that inhibit different steps in the endocytic pathway, including 0.4 M sucrose, ammonium chloride, and brefeldin A, block the acquisition of endo F-resistant sugars and the acquisition of detergent insolubility by newly synthesized anion exchangers. The inhibitory effects of ammonium chloride on anion exchanger processing are rapidly reversible. Furthermore, AE1 anion exchangers become detergent insoluble more rapidly than they acquire endo F-resistant modifications in cells recovering from an ammonium chloride block. This suggests that the cytoskeletal association of the recycling anion exchangers occurs after release from the compartment where they accumulate due to ammonium chloride treatment, and prior to their transit through the Golgi. The recycling pool of newly synthesized anion exchangers is reflected in the steady-state distribution of the polypeptide. In addition to plasma membrane staining, anion exchanger antibodies stain a perinuclear compartment in erythroid cells. This perinuclear AE1-containing compartment is also stained by ankyrin antibodies and partially overlaps the membrane compartment stained by NBD C6-ceramide, a Golgi marker. Detergent extraction of erythroid cells in situ has suggested that a substantial fraction of the perinuclear pool of AE1 is cytoskeletal associated. The demonstration that erythroid anion exchangers interact with elements of the cytoskeleton during recycling to the Golgi suggests the cytoskeleton may be involved in the post-Golgi trafficking of this membrane transporter.

scholarly journals Adaptor protein 2–mediated endocytosis of the β-secretase BACE1 is dispensable for amyloid precursor protein processing

2012 ◽  
Vol 23 (12) ◽  
pp. 2339-2351 ◽  
Author(s):  
Yogikala Prabhu ◽  
Patricia V. Burgos ◽  
Christina Schindler ◽  
Ginny G. Farías ◽  
Javier G. Magadán ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Amyloid Precursor Protein ◽  
Secretory Pathway ◽  
Brefeldin A ◽  
Adaptor Protein ◽  
Proteolytic Processing ◽  
Precursor Protein ◽  
Amyloid Precursor Protein Processing ◽  
Intermediate Compartment ◽  
Golgi Network

The β-site amyloid precursor protein (APP)–cleaving enzyme 1 (BACE1) is a transmembrane aspartyl protease that catalyzes the proteolytic processing of APP and other plasma membrane protein precursors. BACE1 cycles between the trans-Golgi network (TGN), the plasma membrane, and endosomes by virtue of signals contained within its cytosolic C-terminal domain. One of these signals is the DXXLL-motif sequence DISLL, which controls transport between the TGN and endosomes via interaction with GGA proteins. Here we show that the DISLL sequence is embedded within a longer [DE]XXXL[LI]-motif sequence, DDISLL, which mediates internalization from the plasma membrane by interaction with the clathrin-associated, heterotetrameric adaptor protein 2 (AP-2) complex. Mutation of this signal or knockdown of either AP-2 or clathrin decreases endosomal localization and increases plasma membrane localization of BACE1. Remarkably, internalization-defective BACE1 is able to cleave an APP mutant that itself cannot be delivered to endosomes. The drug brefeldin A reversibly prevents BACE1-catalyzed APP cleavage, ruling out that this reaction occurs in the endoplasmic reticulum (ER) or ER–Golgi intermediate compartment. Taken together, these observations support the notion that BACE1 is capable of cleaving APP in late compartments of the secretory pathway.

scholarly journals The unconventional biogenesis of Kv7.1-KCNE1 complexes

2020 ◽  
Vol 6 (14) ◽  
pp. eaay4472 ◽  
Author(s):  
Anna Oliveras ◽  
Clara Serrano-Novillo ◽  
Cristina Moreno ◽  
Alicia de la Cruz ◽  
Carmen Valenzuela ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Plasma Membrane ◽  
Secretory Pathway ◽  
Regulatory Subunit ◽  
Long Qt ◽  
Ongoing Debate ◽  
Subcellular Compartment ◽  
Ongoing Controversy ◽  
The Subject ◽  
Complex Forms

The potassium channel Kv7.1 associates with the KCNE1 regulatory subunit to trigger cardiac IKs currents. Although the Kv7.1/KCNE1 complex has received much attention, the subcellular compartment hosting the assembly is the subject of ongoing debate. Evidence suggests that the complex forms either earlier in the endoplasmic reticulum or directly at the plasma membrane. Kv7.1 and KCNE1 mutations, responsible for long QT syndromes, impair association and traffic, thereby altering IKs currents. We found that Kv7.1 and KCNE1 do not assemble in the first stages of their biogenesis. Data support an unconventional secretory pathway for Kv7.1-KCNE1 that bypasses Golgi. This route targets channels to endoplasmic reticulum–plasma membrane junctions, where Kv7.1-KCNE1 assemble. This mechanism helps to resolve the ongoing controversy about the subcellular compartment hosting the association. Our results also provide new insights into IKs channel localization at endoplasmic reticulum–plasma membrane junctions, highlighting an alternative anterograde trafficking mechanism for oligomeric ion channels.

scholarly journals Palmitoylation and Plasma Membrane Localization of Ras2p by a Nonclassical Trafficking Pathway in Saccharomyces cerevisiae

2003 ◽  
Vol 23 (18) ◽  
pp. 6574-6584 ◽  
Author(s):  
Xiangwen Dong ◽  
David A. Mitchell ◽  
Sandra Lobo ◽  
Lihong Zhao ◽  
Douglas J. Bartels ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Plasma Membrane ◽  
Secretory Pathway ◽  
Brefeldin A ◽  
Covalent Attachment ◽  
Membrane Localization ◽  
Ras Proteins ◽  
Plasma Membrane Localization ◽  
Additional Processing ◽  
Endomembrane Trafficking

ABSTRACT Subcellular localization of Ras proteins to the plasma membrane is accomplished in part by covalent attachment of a farnesyl moiety to the conserved CaaX box cysteine. Farnesylation targets Ras to the endoplasmic reticulum (ER), where additional processing steps occur, resulting in translocation of Ras to the plasma membrane. The mechanism(s) by which this occurs is not well understood. In this report, we show that plasma membrane localization of Ras2p in Saccharomyces cerevisiae does not require the classical secretory pathway or a functional Golgi apparatus. However, when the classical secretory pathway is disrupted, plasma membrane localization requires Erf2p, a protein that resides in the ER membrane and is required for efficient palmitoylation of Ras2p. Deletion of ERF2 results in a Ras2p steady-state localization defect that is more severe when combined with sec-ts mutants or brefeldin A treatment. The Erf2p-dependent localization of Ras2p correlates with the palmitoylation of Cys-318. An Erf2p-Erf4p complex has recently been shown to be an ER-associated palmitoyltransferase that can palmitoylate Cys-318 of Ras2p (S. Lobo, W. K. Greentree, M. E. Linder, and R. J. Deschenes, J. Biol. Chem. 277:41268-41273, 2002). Erf2-dependent palmitoylation as well as localization of Ras2p requires a region of the hypervariable domain adjacent to the CaaX box. These results provide evidence for the existence of a palmitoylation-dependent, nonclassical endomembrane trafficking system for the plasma membrane localization of Ras proteins.

scholarly journals Assembly of influenza hemagglutinin trimers and its role in intracellular transport.

1986 ◽  
Vol 103 (4) ◽  
pp. 1179-1191 ◽  
Author(s):  
C S Copeland ◽  
R W Doms ◽  
E M Bolzau ◽  
R G Webster ◽  
A Helenius
Keyword(s):  
Endoplasmic Reticulum ◽  
Plasma Membrane ◽  
Golgi Complex ◽  
Intracellular Transport ◽  
Secretory Pathway ◽  
Quaternary Structure ◽  
Subunit Interactions ◽  
Oligomer Formation ◽  
Influenza Hemagglutinin ◽  
Triton X

The hemagglutinin (HA) of influenza virus is a homotrimeric integral membrane glycoprotein. It is cotranslationally inserted into the endoplasmic reticulum as a precursor called HA0 and transported to the cell surface via the Golgi complex. We have, in this study, investigated the kinetics and cellular location of the assembly reaction that results in HA0 trimerization. Three independent criteria were used for determining the formation of quaternary structure: the appearance of an epitope recognized by trimer-specific monoclonal antibodies; the acquisition of trypsin resistance, a characteristic of trimers; and the formation of stable complexes which cosedimented with the mature HA0 trimer (9S20,w) in sucrose gradients containing Triton X-100. The results showed that oligomer formation is a posttranslational event, occurring with a half time of approximately 7.5 min after completion of synthesis. Assembly occurs in the endoplasmic reticulum, followed almost immediately by transport to the Golgi complex. A stabilization event in trimer structure occurs when HA0 leaves the Golgi complex or reaches the plasma membrane. Approximately 10% of the newly synthesized HA0 formed aberrant trimers which were not transported from the endoplasmic reticulum to the Golgi complex or the plasma membrane. Taken together the results suggested that formation of correctly folded quaternary structure constitutes a key event regulating the transport of the protein out of the endoplasmic reticulum. Further changes in subunit interactions occur as the trimers move along the secretory pathway.

Leptin biosynthetic pathway in white adipocytes

2006 ◽  
Vol 84 (2) ◽  
pp. 207-214 ◽  
Author(s):  
Philippe G Cammisotto ◽  
Ludwik J Bukowiecki ◽  
Yves Deshaies ◽  
Moise Bendayan
Keyword(s):  
Endoplasmic Reticulum ◽  
Rough Endoplasmic Reticulum ◽  
Secretory Pathway ◽  
De Novo ◽  
Brefeldin A ◽  
Mrna Levels ◽  
De Novo Synthesis ◽  
Cellular Content ◽  
Constitutive Secretion

The aim of this study was to determine through morphological and biochemical means the biosynthetic and secretory pathway followed by leptin in adipocytes. Immunocytochemistry revealed the presence of leptin in the rough endoplasmic reticulum, the Golgi apparatus, and in numerous small vesicles along the plasma membrane of white adipo cytes. In vitro, isolated adipocytes under nonstimulated conditions (basal) continuously secreted leptin while their intra cellular content remained unchanged. When adipocytes were stimulated with insulin, leptin cellular content and secretion increased in parallel and were significantly different from basal secretion only after 45 min. L-leucine and L-glutamate also strongly stimulated leptin synthesis and secretion. These stimulating effects were abolished by cycloheximide and brefeldin A. The transcriptional inhibitor actinomycin D did not have any effects in either basal or stimulated conditions. Leptin mRNA levels were not affected by any stimulating or inhibiting agents. Finally, norepinephrine, isoproterenol, CL316243, and palmitate inhibited the effects of insulin, L-leucine, and L-glutamate on leptin synthesis. We thus conclude that (i) adipocytes continuously synthesize and secrete leptin along a rough endoplasmic reticulum–Golgi secretory vesicles pathway, (ii) an increase in leptin secretion requires increased de novo synthesis, and (iii) short-term leptin secretion does not involve changes in mRNA levels.Key words: leptin, vesicles, constitutive secretion, de novo synthesis, transcription.

scholarly journals Transmembrane domain–dependent partitioning of membrane proteins within the endoplasmic reticulum

2008 ◽  
Vol 181 (1) ◽  
pp. 105-118 ◽  
Author(s):  
Paolo Ronchi ◽  
Sara Colombo ◽  
Maura Francolini ◽  
Nica Borgese
Keyword(s):  
Endoplasmic Reticulum ◽  
Plasma Membrane ◽  
Membrane Proteins ◽  
Secretory Pathway ◽  
Transmembrane Domain ◽  
Fluorescent Proteins ◽  
Lipid Interactions ◽  
Er Exit Sites ◽  
Physicochemical Features ◽  
Er Membrane

The length and hydrophobicity of the transmembrane domain (TMD) play an important role in the sorting of membrane proteins within the secretory pathway; however, the relative contributions of protein–protein and protein–lipid interactions to this phenomenon are currently not understood. To investigate the mechanism of TMD-dependent sorting, we used the following two C tail–anchored fluorescent proteins (FPs), which differ only in TMD length: FP-17, which is anchored to the endoplasmic reticulum (ER) membrane by 17 uncharged residues, and FP-22, which is driven to the plasma membrane by its 22-residue-long TMD. Before export of FP-22, the two constructs, although freely diffusible, were seen to distribute differently between ER tubules and sheets. Analyses in temperature-blocked cells revealed that FP-17 is excluded from ER exit sites, whereas FP-22 is recruited to them, although it remains freely exchangeable with the surrounding reticulum. Thus, physicochemical features of the TMD influence sorting of membrane proteins both within the ER and at the ER–Golgi boundary by simple receptor-independent mechanisms based on partitioning.

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