The unconventional biogenesis of Kv7.1-KCNE1 complexes

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.

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Life and Death of Fungal Transporters Under the Challenge of Polarity

10.20944/preprints202007.0601.v1 ◽

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.

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

The Journal of Cell Biology ◽

10.1083/jcb.103.4.1179 ◽

1986 ◽

Vol 103 (4) ◽

pp. 1179-1191 ◽

Cited By ~ 243

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.

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

The Journal of Cell Biology ◽

10.1083/jcb.200710093 ◽

2008 ◽

Vol 181 (1) ◽

pp. 105-118 ◽

Cited By ~ 65

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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Visualizing Wnt secretion from Endoplasmic Reticulum to Filopodia

10.1101/271684 ◽

2018 ◽

Author(s):

Naushad Moti ◽

Jia Yu ◽

Gaelle Boncompain ◽

Franck Perez ◽

David M Virshup

Keyword(s):

Endoplasmic Reticulum ◽

Plasma Membrane ◽

Secretory Pathway ◽

Signaling Cascades ◽

Downstream Signaling ◽

Cell Compartments ◽

A Cell ◽

Cortical Regions ◽

Rate Limiting ◽

Wnt Signal

AbstractWnts are a family of secreted palmitoleated glycoproteins that play a key role in cell to cell communications during development and regulate stem cell compartments in adults. Wnt receptors, downstream signaling cascades and target pathways have been extensively studied while less is known about how Wnts are secreted and move from producing cells to receiving cells. We used the synchronization system called Retention Using Selective Hook (RUSH) to study Wnt trafficking from endoplasmic reticulum to Golgi and then to plasma membrane and filopodia in real time. Consistent with prior studies, inhibition of porcupine (PORCN) or knockout of Wntless (WLS) blocked Wnt exit from the ER. Indeed, WLS was rate-limiting for Wnt ER exit. Wnt-containing vesicles paused at sub-cortical regions of the plasma membrane before exiting the cell. Wnt-containing vesicles were transported to adjacent cells associated with filopodia. Increasing the number of filopodia by expression of LGR5 in the producing cell increased the ability of a cell to send a Wnt signal. The RUSH system is a powerful tool to provide new insights into the Wnt secretory pathway.

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The G protein–coupled receptor GPR31 promotes membrane association of KRAS

The Journal of Cell Biology ◽

10.1083/jcb.201609096 ◽

2017 ◽

Vol 216 (8) ◽

pp. 2329-2338 ◽

Cited By ~ 9

Author(s):

Nicole Fehrenbacher ◽

Israel Tojal da Silva ◽

Craig Ramirez ◽

Yong Zhou ◽

Kwang-Jin Cho ◽

...

Keyword(s):

Endoplasmic Reticulum ◽

Plasma Membrane ◽

Tumor Cells ◽

G Protein ◽

Secretory Pathway ◽

Membrane Association ◽

G Protein Coupled Receptor ◽

Genome Wide ◽

A Genome ◽

G Protein Coupled

The product of the KRAS oncogene, KRAS4B, promotes tumor growth when associated with the plasma membrane (PM). PM association is mediated, in part, by farnesylation of KRAS4B, but trafficking of nascent KRAS4B to the PM is incompletely understood. We performed a genome-wide screen to identify genes required for KRAS4B membrane association and identified a G protein–coupled receptor, GPR31. GPR31 associated with KRAS4B on cellular membranes in a farnesylation-dependent fashion, and retention of GPR31 on the endoplasmic reticulum inhibited delivery of KRAS4B to the PM. Silencing of GPR31 expression partially mislocalized KRAS4B, slowed the growth of KRAS-dependent tumor cells, and blocked KRAS-stimulated macropinocytosis. Our data suggest that GPR31 acts as a secretory pathway chaperone for KRAS4B.

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Life and Death of Fungal Transporters under the Challenge of Polarity

International Journal of Molecular Sciences ◽

10.3390/ijms21155376 ◽

2020 ◽

Vol 21 (15) ◽

pp. 5376

Author(s):

Sofia Dimou ◽

George Diallinas

Keyword(s):

Endoplasmic Reticulum ◽

Plasma Membrane ◽

Cell Polarity ◽

Secretory Pathway ◽

Fungal Growth ◽

Multivesicular Bodies ◽

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 multivesicular bodies (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 of why transporters and possibly other housekeeping membrane proteins ‘avoid’ routes of polar trafficking.

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Crosstalk between Endoplasmic Reticulum Stress and Protein Misfolding in Neurodegenerative Diseases

ISRN Cell Biology ◽

10.1155/2013/256404 ◽

2013 ◽

Vol 2013 ◽

pp. 1-22 ◽

Cited By ~ 27

Author(s):

Cláudia M. F. Pereira

Keyword(s):

Quality Control ◽

Endoplasmic Reticulum ◽

Neurodegenerative Diseases ◽

Protein Misfolding ◽

Secretory Pathway ◽

Protein Quality ◽

Prion Diseases ◽

Protein Quality Control ◽

Subcellular Compartment ◽

Novel Therapeutic Strategies

Under physiological conditions, the endoplasmic reticulum (ER) is a central subcellular compartment for protein quality control in the secretory pathway that prevents protein misfolding and aggregation. Instrumental in protein quality control in the ER is the unfolded protein response (UPR), which is activated upon ER stress to reestablish homeostasis through a sophisticated transcriptionally and translationally regulated signaling network. However, this response can lead to apoptosis if the stress cannot be alleviated. The presence of abnormal protein aggregates containing specific misfolded proteins is recognized as the basis of numerous human conformational disorders, including neurodegenerative diseases. Here, I will highlight the overwhelming evidence that the presence of specific aberrant proteins in Alzheimer’s disease (AD), Parkinson’s disease (PD), Huntington’s disease (HD), prion diseases, and Amyotrophic Lateral Sclerosis (ALS) is intimately associated with perturbations in the ER protein quality control machinery that become incompetent to restore protein homeostasis and shift adaptive programs toward the induction of apoptotic signaling to eliminate irreversibly damaged neurons. Increasing our understanding about the deadly crosstalk between ER dysfunction and protein misfolding in these neurodegenerative diseases may stimulate the development of novel therapeutic strategies able to support neuronal survival and ameliorate disease progression.

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COOH-terminal signals mediate the trafficking of a peptide processing enzyme in endocrine cells.

The Journal of Cell Biology ◽

10.1083/jcb.121.1.23 ◽

1993 ◽

Vol 121 (1) ◽

pp. 23-36 ◽

Cited By ~ 50

Author(s):

S L Milgram ◽

R E Mains ◽

B A Eipper

Keyword(s):

Plasma Membrane ◽

Cell Surface ◽

Endocrine Cells ◽

Secretory Pathway ◽

Bioactive Peptides ◽

Full Length ◽

Neuroendocrine Cells ◽

Subcellular Compartment ◽

Peptide Processing ◽

Regulated Secretory Pathway

Peptidylglycine alpha-amidating monooxygenase (PAM) catalyzes the COOH-terminal amidation of bioactive peptides through a two step reaction catalyzed by separate enzymes contained within the PAM precursor. To characterize the trafficking of integral membrane PAM proteins in neuroendocrine cells, we have generated stable AtT-20 cell lines expressing full length and COOH-terminally truncated integral membrane PAM proteins. Full length integral membrane PAM was present on the cell surface in low but detectable amounts and PAM proteins which reached the cell surface were rapidly internalized but not immediately degraded in lysosomes. Internalized PAM complexed with PAM antibody was found in a subcellular compartment which overlapped with internalized transferrin and with structures binding WGA. Thus the punctate juxtanuclear staining of full length PAM represents PAM in endosomes. Endoproteolytic processing of full length PAM-1 and PAM-2 resulted in the secretion of soluble PAM proteins; the secretion of these soluble PAM proteins was stimulus dependent. Although some of the truncated PAM protein was also processed and stored in AtT-20 cells, much of the expressed protein was redistributed to the plasma membrane. Soluble proteins not observed in large amounts in cells expressing full length PAM were released from the surface of cells expressing truncated PAM and little internalization of truncated integral membrane PAM was observed. Thus, the COOH-terminal domain of PAM contains information important for its trafficking within the regulated secretory pathway as well as information necessary for its retrieval from the cell surface.

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Erf4p and Erf2p Form an Endoplasmic Reticulum-associated Complex Involved in the Plasma Membrane Localization of Yeast Ras Proteins

Journal of Biological Chemistry ◽

10.1074/jbc.m209760200 ◽

2002 ◽

Vol 277 (51) ◽

pp. 49352-49359 ◽

Cited By ~ 62

Author(s):

Lihong Zhao ◽

Sandra Lobo ◽

Xiangwen Dong ◽

Addison D. Ault ◽

Robert J. Deschenes

Keyword(s):

Endoplasmic Reticulum ◽

Plasma Membrane ◽

Membrane Protein ◽

Secretory Pathway ◽

Integral Membrane Protein ◽

Genetic Screen ◽

Membrane Localization ◽

Ras Proteins ◽

Transport Pathway ◽

Plasma Membrane Localization

Ras oncogene proteins are plasma membrane-associated signal transducers that are found in all eukaryotes. Posttranslational addition of lipid to a carboxyl-terminal CaaXbox (where “C” represents a cysteine, “a” is generally an aliphatic residue, andXcan be any amino acid) is required to target Ras proteins to the cytosolic surface of the plasma membrane. The pathway by which Ras translocates from the endoplasmic reticulum to the plasma membrane is currently not clear. We have performed a genetic screen to identify components of the Ras plasma membrane localization pathway. Mutations in two genes,ERF2andERF4/SHR5, have been shown to affect the palmitoylation and subcellular localization of Ras proteins. In this report, we show that Erf4p is localized on the endoplasmic reticulum as a peripheral membrane protein in a complex with Erf2p, an integral membrane protein that was identified from the same genetic screen. Erf2p has been shown to be required for the plasma membrane localization of GFP-Ras2p via a pathway distinct from the classical secretory pathway (X. Dong and R. J. Deschenes, manuscript in preparation). We show here that Erf4p, like Erf2p, is involved in the plasma membrane localization of Ras2p. Erf2p and Erf4p represent components of a previously uncharacterized subcellular transport pathway involved in the plasma membrane targeting of Ras proteins.

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

10.1101/744177 ◽

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.

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