Ykt6 membrane-to-cytosol cycling regulates exosomal Wnt secretion

Protein trafficking in the secretory pathway, for example the secretion of Wnt proteins, requires tight regulation. These ligands activate Wnt signaling pathways and are crucially involved in development and disease. Wnt is transported to the plasma membrane by its cargo receptor Evi, where Wnt/Evi complexes are endocytosed and sorted onto exosomes for long-range secretion. However, the trafficking steps within the endosomal compartment are not fully understood. The promiscuous SNARE Ykt6 folds into an auto-inhibiting conformation in the cytosol, but a portion associates with membranes by its farnesylated and palmitoylated C-terminus. Here, we demonstrate that membrane detachment of Ykt6 is essential for exosomal Wnt secretion. We identified conserved phosphorylation sites within the SNARE domain of Ykt6, which block Ykt6 cycling from the membrane to the cytosol. In Drosophila, Ykt6-RNAi mediated block of Wg secretion is rescued by wildtype but not phosphomimicking Ykt6. The latter accumulates at membranes, while wildtype Ykt6 regulates Wnt trafficking between the plasma membrane and multivesicular bodies. Taken together, we show that a regulatory switch in Ykt6 fine-tunes sorting of Wnts in endosomes.

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Misregulation of Wnt Signaling Pathways at the Plasma Membrane in Brain and Metabolic Diseases

Membranes ◽

10.3390/membranes11110844 ◽

2021 ◽

Vol 11 (11) ◽

pp. 844

Author(s):

Mustafa Karabicici ◽

Yagmur Azbazdar ◽

Evin Iscan ◽

Gunes Ozhan

Keyword(s):

Plasma Membrane ◽

Wnt Signaling ◽

Signaling Pathways ◽

Metabolic Diseases ◽

Wnt Signaling Pathway ◽

Membrane Domains ◽

Physiological Processes ◽

Pathway Activation ◽

Wnt Pathways ◽

Membrane Components

Wnt signaling pathways constitute a group of signal transduction pathways that direct many physiological processes, such as development, growth, and differentiation. Dysregulation of these pathways is thus associated with many pathological processes, including neurodegenerative diseases, metabolic disorders, and cancer. At the same time, alterations are observed in plasma membrane compositions, lipid organizations, and ordered membrane domains in brain and metabolic diseases that are associated with Wnt signaling pathway activation. Here, we discuss the relationships between plasma membrane components—specifically ligands, (co) receptors, and extracellular or membrane-associated modulators—to activate Wnt pathways in several brain and metabolic diseases. Thus, the Wnt–receptor complex can be targeted based on the composition and organization of the plasma membrane, in order to develop effective targeted therapy drugs.

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Crosstalk between autophagy inhibitors and endosome-related secretory pathways: a challenge for autophagy-based treatment of solid cancers

Molecular Cancer ◽

10.1186/s12943-021-01423-6 ◽

2021 ◽

Vol 20 (1) ◽

Author(s):

Martina Raudenska ◽

Jan Balvan ◽

Michal Masarik

Keyword(s):

Cancer Progression ◽

Protein Trafficking ◽

Secretory Pathway ◽

Clinical Results ◽

Multivesicular Bodies ◽

Cancer Type ◽

Secretory Pathways ◽

Specific Effects ◽

Antineoplastic Treatment ◽

Autophagy Inhibition

AbstractAutophagy is best known for its role in organelle and protein turnover, cell quality control, and metabolism. The autophagic machinery has, however, also adapted to enable protein trafficking and unconventional secretory pathways so that organelles (such as autophagosomes and multivesicular bodies) delivering cargo to lysosomes for degradation can change their mission from fusion with lysosomes to fusion with the plasma membrane, followed by secretion of the cargo from the cell. Some factors with key signalling functions do not enter the conventional secretory pathway but can be secreted in an autophagy-mediated manner.Positive clinical results of some autophagy inhibitors are encouraging. Nevertheless, it is becoming clear that autophagy inhibition, even within the same cancer type, can affect cancer progression differently. Even next-generation inhibitors of autophagy can have significant non-specific effects, such as impacts on endosome-related secretory pathways and secretion of extracellular vesicles (EVs). Many studies suggest that cancer cells release higher amounts of EVs compared to non-malignant cells, which makes the effect of autophagy inhibitors on EVs secretion highly important and attractive for anticancer therapy. In this review article, we discuss how different inhibitors of autophagy may influence the secretion of EVs and summarize the non-specific effects of autophagy inhibitors with a focus on endosome-related secretory pathways. Modulation of autophagy significantly impacts not only the quantity of EVs but also their content, which can have a deep impact on the resulting pro-tumourigenic or anticancer effect of autophagy inhibitors used in the antineoplastic treatment of solid cancers.

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Role of the Transmembrane Domain of Marburg Virus Surface Protein GP in Assembly of the Viral Envelope

Journal of Virology ◽

10.1128/jvi.02263-06 ◽

2007 ◽

Vol 81 (8) ◽

pp. 3942-3948 ◽

Cited By ~ 30

Author(s):

Eva Mittler ◽

Larissa Kolesnikova ◽

Thomas Strecker ◽

Wolfgang Garten ◽

Stephan Becker

Keyword(s):

Plasma Membrane ◽

Secretory Pathway ◽

Matrix Protein ◽

Transmembrane Domain ◽

Transmembrane Protein ◽

Marburg Virus ◽

Viral Envelope ◽

Major Protein ◽

Multivesicular Bodies ◽

Virus Surface

ABSTRACT The major protein constituents of the filoviral envelope are the matrix protein VP40 and the surface transmembrane protein GP. While VP40 is recruited to the sites of budding via the late retrograde endosomal transport route, GP is suggested to be transported via the classical secretory pathway involving the endoplasmic reticulum, Golgi apparatus, and trans-Golgi network until it reaches the plasma membrane where most filoviral budding takes place. Since both transport routes target the plasma membrane, it was thought that GP and VP40 join there to form the viral envelope. However, it was recently shown that, upon coexpression of both proteins, GP is partially recruited into peripheral VP40-enriched multivesicular bodies, which contained markers of the late endosome. Accumulation of GP and VP40 in this compartment was presumed to play an important role in the formation of the filoviral envelope. Using a domain-swapping approach, we were able to show that the transmembrane domain of GP was essential and sufficient for (i) partial recruitment of chimeric glycoproteins into VP40-enriched multivesicular bodies and (ii) incorporation into virus-like particles (VLPs) that were released upon expression of VP40. Only those chimeric glycoproteins which were targeted to VP40-enriched endosomal multivesicular bodies were subsequently recruited into VLPs. These data show that the transmembrane domain of GP is critical for the mixing of VP40 and GP in multivesicular bodies and incorporation of GP into the viral envelope. Results further suggest that trapping of GP in the VP40-enriched late endosomal compartment is important for the formation of the viral envelope.

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C Terminus of Nce102 Determines the Structure and Function of Microdomains in the Saccharomyces cerevisiae Plasma Membrane

Eukaryotic Cell ◽

10.1128/ec.00006-10 ◽

2010 ◽

Vol 9 (8) ◽

pp. 1184-1192 ◽

Cited By ~ 24

Author(s):

Martin Loibl ◽

Guido Grossmann ◽

Vendula Stradalova ◽

Andreas Klingl ◽

Reinhard Rachel ◽

...

Keyword(s):

Saccharomyces Cerevisiae ◽

Plasma Membrane ◽

Protein Trafficking ◽

Ultrastructural Feature ◽

Membrane Topology ◽

Content Type ◽

C Terminus ◽

Composition And Structure ◽

And Function ◽

Arginine Permease

ABSTRACT The plasma membrane of the yeast Saccharomyces cerevisiae contains stably distributed lateral domains of specific composition and structure, termed MCC (membrane compartment of arginine permease Can1). Accumulation of Can1 and other specific proton symporters within MCC is known to regulate the turnover of these transporters and is controlled by the presence of another MCC protein, Nce102. We show that in an NCE102 deletion strain the function of Nce102 in directing the specific permeases into MCC can be complemented by overexpression of the NCE102 close homolog FHN1 (the previously uncharacterized YGR131W) as well as by distant Schizosaccharomyces pombe homolog fhn1 (SPBC1685.13). We conclude that this mechanism of plasma membrane organization is conserved through the phylum Ascomycota. We used a hemagglutinin (HA)/Suc2/His4C reporter to determine the membrane topology of Nce102. In contrast to predictions, its N and C termini are oriented toward the cytosol. Deletion of the C terminus or even of its last 6 amino acids does not disturb protein trafficking, but it seriously affects the formation of MCC. We show that the C-terminal part of the Nce102 protein is necessary for localization of both Nce102 itself and Can1 to MCC and also for the formation of furrow-like membrane invaginations, the characteristic ultrastructural feature of MCC domains.

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Homo- and hetero-dimeric architecture of the human liver Na+-dependent taurocholate co-transporting protein

Biochemical Journal ◽

10.1042/bj20111234 ◽

2012 ◽

Vol 441 (3) ◽

pp. 1007-1016 ◽

Cited By ~ 37

Author(s):

Ingrid T. G. W. Bijsmans ◽

Rianne A. M. Bouwmeester ◽

Joachim Geyer ◽

Klaas Nico Faber ◽

Stan F. J. van de Graaf

Keyword(s):

Plasma Membrane ◽

Bile Salt ◽

Membrane Trafficking ◽

Secretory Pathway ◽

Physical Interaction ◽

Oligomeric State ◽

Salt Uptake ◽

Transporter Family ◽

C Terminus ◽

U2os Cells

The NTCP (Na+–taurocholate co-transporting protein)/SLC10A [solute carrier family 10 (Na+/bile acid co-transporter family)] 1 is tightly controlled to ensure hepatic bile salt uptake while preventing toxic bile salt accumulation. Many transport proteins require oligomerization for their activity and regulation. This is not yet established for bile salt transporters. The present study was conducted to elucidate the oligomeric state of NTCP. Chemical cross-linking revealed the presence of NTCP dimers in rat liver membranes and U2OS cells stably expressing NTCP. Co-immunoprecipitation of tagged NTCP proteins revealed a physical interaction between subunits. The C-terminus of NTCP was not required for subunit interaction, but was essential for exit from the ER (endoplasmic reticulum). NTCP without its C-terminus (NTCP Y307X) retained full-length wtNTCP (wild-type NTCP) in the ER in a dominant fashion, suggesting that dimerization occurs early in the secretory pathway. FRET (fluorescence resonance energy transfer) using fluorescently labelled subunits further demonstrated that dimerization persists at the plasma membrane. NTCP belongs to the SLC10A protein family which consists of seven members. NTCP co-localized in U2OS cells with SLC10A4 and SLC10A6, but not with SLC10A3, SLC10A5 or SLC10A7. SLC10A4 and SLC10A6 co-immunoprecipitated with NTCP, demonstrating that heteromeric complexes can be formed between SLC10A family members in vitro. Expression of SLC10A4 and NTCP Y307X resulted in a reduction of NTCP abundance at the plasma membrane and NTCP-mediated taurocholate uptake, whereas expression of SLC10A6 or NTCP E257N, an inactive mutant, did not affect NTCP function. In conclusion, NTCP adopts a dimeric structure in which individual subunits are functional. Bile salt uptake is influenced by heterodimerization when this impairs NTCP plasma membrane trafficking.

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Subcellular Localization and Topology of the p7 Polypeptide of Hepatitis C Virus

Journal of Virology ◽

10.1128/jvi.76.8.3720-3730.2002 ◽

2002 ◽

Vol 76 (8) ◽

pp. 3720-3730 ◽

Cited By ~ 143

Author(s):

Séverine Carrère-Kremer ◽

Claire Montpellier-Pala ◽

Laurence Cocquerel ◽

Czeslaw Wychowski ◽

François Penin ◽

...

Keyword(s):

Plasma Membrane ◽

Hepatitis C Virus ◽

Hepatitis C ◽

Subcellular Localization ◽

Secretory Pathway ◽

Transmembrane Domain ◽

Signal Sequence ◽

C Terminus ◽

Membrane Spanning ◽

Polytopic Membrane Protein

ABSTRACT Although biological and biochemical data have been accumulated on most hepatitis C virus proteins, the structure and function of the 63-amino-acid p7 polypeptide of this virus have never been investigated. In this work, sequence analyses predicted that p7 contains two transmembrane passages connected by a short hydrophilic segment. The C-terminal transmembrane domain of p7 was predicted to function as a signal sequence, which was confirmed experimentally by analyzing the translocation of a reporter glycoprotein fused at its C terminus. The p7 polypeptide was tagged either with the ectodomain of CD4 or with a Myc epitope to study its membrane integration, its subcellular localization, and its topology. Alkaline extraction studies confirmed that p7 is an integral membrane polypeptide. The CD4-p7 chimera was detected by immunofluorescence on the surface of nonpermeabilized cells, indicating that it is exported to the plasma membrane. However, pulse-chase analyses showed that only approximately 20% of endoglycosidase H-resistant CD4-p7 was detected after long chase times, suggesting that a large proportion of p7 stays in an early compartment of the secretory pathway. Finally, by inserting a Myc epitope in several positions of p7 and analyzing the accessibility of this epitope on the plasma membrane of HepG2 cells, we showed that p7 has a double membrane-spanning topology, with both its N and C termini oriented toward the extracellular environment. Altogether, these data indicate that p7 is a polytopic membrane protein that could have a functional role in several compartments of the secretory pathway.

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Regulation of Wnt Signaling Pathways at the Plasma Membrane and Their Misregulation in Cancer

Frontiers in Cell and Developmental Biology ◽

10.3389/fcell.2021.631623 ◽

2021 ◽

Vol 9 ◽

Author(s):

Yagmur Azbazdar ◽

Mustafa Karabicici ◽

Esra Erdal ◽

Gunes Ozhan

Keyword(s):

Plasma Membrane ◽

Wnt Signaling ◽

Signaling Pathways ◽

Cancer Progression ◽

Malignant Tumors ◽

Wnt Signaling Pathway ◽

Receptor Complex ◽

Breast Cancers ◽

Membrane Composition ◽

Membrane Components

Wnt signaling is one of the key signaling pathways that govern numerous physiological activities such as growth, differentiation and migration during development and homeostasis. As pathway misregulation has been extensively linked to pathological processes including malignant tumors, a thorough understanding of pathway regulation is essential for development of effective therapeutic approaches. A prominent feature of cancer cells is that they significantly differ from healthy cells with respect to their plasma membrane composition and lipid organization. Here, we review the key role of membrane composition and lipid order in activation of Wnt signaling pathway by tightly regulating formation and interactions of the Wnt-receptor complex. We also discuss in detail how plasma membrane components, in particular the ligands, (co)receptors and extracellular or membrane-bound modulators, of Wnt pathways are affected in lung, colorectal, liver and breast cancers that have been associated with abnormal activation of Wnt signaling. Wnt-receptor complex components and their modulators are frequently misexpressed in these cancers and this appears to correlate with metastasis and cancer progression. Thus, composition and organization of the plasma membrane can be exploited to develop new anticancer drugs that are targeted in a highly specific manner to the Wnt-receptor complex, rendering a more effective therapeutic outcome possible.

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Ezrin: a protein requiring conformational activation to link microfilaments to the plasma membrane in the assembly of cell surface structures

Journal of Cell Science ◽

10.1242/jcs.110.24.3011 ◽

1997 ◽

Vol 110 (24) ◽

pp. 3011-3018 ◽

Cited By ~ 21

Author(s):

A. Bretscher ◽

D. Reczek ◽

M. Berryman

Keyword(s):

Plasma Membrane ◽

Signaling Pathways ◽

Regulatory Subunit ◽

Membrane Domains ◽

Structural Support ◽

Binding Partners ◽

Cell Surface Structures ◽

C Terminus ◽

Biological Studies ◽

Cortical Cytoskeleton

The cortical cytoskeleton of eucaryotic cells provides structural support to the plasma membrane and also contributes to dynamic processes such as endocytosis, exocytosis, and transmembrane signaling pathways. The ERM (ezrin-radixin-moesin) family of proteins, of which ezrin is the best studied member, play structural and regulatory roles in the assembly and stabilization of specialized plasma membrane domains. Ezrin and related molecules are concentrated in surface projections such as microvilli and membrane ruffles where they link the microfilaments to the membrane. The present knowledge about ezrin is discussed from an historical perspective. Both biochemical and cell biological studies have revealed that ezrin can exist in a dormant conformation that requires activation to expose otherwise masked association sites. Current results indicate that activated ezrin monomers or head-to-tail oligomers associate directly with F-actin through a domain in its C terminus, and with the membrane through its N-terminal domain. The association of ezrin with transmembrane proteins can be direct, as in the case of CD44, or indirect through EBP50. Other binding partners, including the regulatory subunit of protein kinase A and rho-GDI, suggest that ezrin is an integral component of these signaling pathways. Although the membrane-cytoskeletal linking function is clear, further studies are necessary to reveal how the activation of ezrin and its association with different binding partners is regulated.

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WNT3a and WNT5a Transported by Exosomes Activate WNT Signaling Pathways in Human Cardiac Fibroblasts

International Journal of Molecular Sciences ◽

10.3390/ijms20061436 ◽

2019 ◽

Vol 20 (6) ◽

pp. 1436 ◽

Cited By ~ 13

Author(s):

Edyta Działo ◽

Michał Rudnik ◽

Roman Koning ◽

Marcin Czepiel ◽

Karolina Tkacz ◽

...

Keyword(s):

Wnt Signaling ◽

Signaling Pathways ◽

Cardiac Fibrosis ◽

Nuclear Translocation ◽

Glycogen Synthase ◽

Cardiac Fibroblasts ◽

Interacting Protein ◽

L Cells ◽

Wnt Proteins ◽

Human Cardiac Fibroblasts

WNT signaling plays an important role in fibrotic processes in the heart. Recently, exosomes have been proposed as novel extracellular transporters for WNT proteins. In this study, we analyzed whether WNT3a and WNT5a carried by exosomes could activate downstream molecular pathways in human cardiac fibroblasts. Exosomes were isolated from conditioned medium of control, WNT3a- and WNT5a-producing L cells by differential ultracentrifugations. Obtained exosomes showed size ranging between 20–150 nm and expressed exosomal markers ALG-2-interacting protein X (ALIX) and CD63. Treatment with WNT3a-rich exosomes inhibited activity of glycogen synthase kinase 3β (GSK3β), induced nuclear translocation of β-catenin, and activated T-cell factor (TCF)/lymphoid enhancer factor (LEF) transcription factors as well as expression of WNT/β-catenin responsive genes in cardiac fibroblasts, but did not coactivate extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK), and activator protein 1 (AP-1) signaling pathways. In contrast, exosomes produced by WNT5a-producing L cells failed to activate β-catenin-dependent response, but successfully triggered phosphorylation of ERK1/2 and JNK and stimulated IL-6 production. In conclusion, exosomes containing WNT proteins can functionally contribute to cardiac fibrosis by activating profibrotic WNT pathways on cardiac fibroblasts and may represent a novel mechanism of spreading profibrotic signals in the heart.

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Sclerostin inhibits Wnt signaling through tandem interaction with two LRP6 ectodomains

Nature Communications ◽

10.1038/s41467-020-19155-4 ◽

2020 ◽

Vol 11 (1) ◽

Author(s):

Jinuk Kim ◽

Wonhee Han ◽

Taeyong Park ◽

Eun Jin Kim ◽

Injin Bang ◽

...

Keyword(s):

Wnt Signaling ◽

Low Density Lipoprotein ◽

Wnt Signaling Pathway ◽

Density Lipoprotein ◽

Interaction Sites ◽

Wnt Proteins ◽

C Terminus ◽

Density Lipoprotein Receptor

Abstract Low-density lipoprotein receptor-related protein 6 (LRP6) is a coreceptor of the β-catenin-dependent Wnt signaling pathway. The LRP6 ectodomain binds Wnt proteins, as well as Wnt inhibitors such as sclerostin (SOST), which negatively regulates Wnt signaling in osteocytes. Although LRP6 ectodomain 1 (E1) is known to interact with SOST, several unresolved questions remain, such as the reason why SOST binds to LRP6 E1E2 with higher affinity than to the E1 domain alone. Here, we present the crystal structure of the LRP6 E1E2–SOST complex with two interaction sites in tandem. The unexpected additional binding site was identified between the C-terminus of SOST and the LRP6 E2 domain. This interaction was confirmed by in vitro binding and cell-based signaling assays. Its functional significance was further demonstrated in vivo using Xenopus laevis embryos. Our results provide insights into the inhibitory mechanism of SOST on Wnt signaling.

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