scholarly journals Lateral transport of Smoothened from the plasma membrane to the membrane of the cilium

2009 ◽  
Vol 187 (3) ◽  
pp. 365-374 ◽  
Author(s):  
Ljiljana Milenkovic ◽  
Matthew P. Scott ◽  
Rajat Rohatgi
Keyword(s):  
Plasma Membrane ◽  
Protein Trafficking ◽  
Primary Cilia ◽  
Protein Transport ◽  
Transmembrane Protein ◽  
Signaling Proteins ◽  
Lateral Transport ◽  
Transport Pathway ◽  
Ciliary Membrane ◽  
Specific Proteins

The function of primary cilia depends critically on the localization of specific proteins in the ciliary membrane. A major challenge in the field is to understand protein trafficking to cilia. The Hedgehog (Hh) pathway protein Smoothened (Smo), a 7-pass transmembrane protein, moves to cilia when a ligand is received. Using microscopy-based pulse-chase analysis, we find that Smo moves through a lateral transport pathway from the plasma membrane to the ciliary membrane. Lateral movement, either via diffusion or active transport, is quite distinct from currently studied pathways of ciliary protein transport in mammals, which emphasize directed trafficking of Golgi-derived vesicles to the base of the cilium. We anticipate that this alternative route will be used by other signaling proteins that function at cilia. The path taken by Smo may allow novel strategies for modulation of Hh signaling in cancer and regeneration.

scholarly journals BLOC-1 is required for selective membrane protein trafficking from endosomes to primary cilia

2017 ◽  
Vol 216 (7) ◽  
pp. 2131-2150 ◽  
Author(s):  
William J. Monis ◽  
Victor Faundez ◽  
Gregory J. Pazour
Keyword(s):  
Protein Trafficking ◽  
Primary Cilia ◽  
Recycling Endosome ◽  
Ciliary Membrane ◽  
Membrane Protein Trafficking ◽  
Selective Membrane ◽  
Specific Proteins ◽  
Lysosome Related Organelles ◽  
Extracellular Environment

Primary cilia perceive the extracellular environment through receptors localized in the ciliary membrane, but mechanisms directing specific proteins to this domain are poorly understood. To address this question, we knocked down proteins potentially important for ciliary membrane targeting and determined how this affects the ciliary trafficking of fibrocystin, polycystin-2, and smoothened. Our analysis showed that fibrocystin and polycystin-2 are dependent on IFT20, GMAP210, and the exocyst complex, while smoothened delivery is largely independent of these components. In addition, we found that polycystin-2, but not smoothened or fibrocystin, requires the biogenesis of lysosome-related organelles complex-1 (BLOC-1) for ciliary delivery. Consistent with the role of BLOC-1 in sorting from the endosome, we find that disrupting the recycling endosome reduces ciliary polycystin-2 and causes its accumulation in the recycling endosome. This is the first demonstration of a role for BLOC-1 in ciliary assembly and highlights the complexity of pathways taken to the cilium.

Molecular basis of ciliary defects caused by compound heterozygous IFT144/WDR19 mutations found in cranioectodermal dysplasia

2021 ◽  
Author(s):  
Yamato Ishida ◽  
Takuya Kobayashi ◽  
Shuhei Chiba ◽  
Yohei Katoh ◽  
Kazuhisa Nakayama
Keyword(s):  
Protein Trafficking ◽  
Primary Cilia ◽  
Intraflagellar Transport ◽  
Missense Variant ◽  
Cellular Level ◽  
Compound Heterozygous ◽  
Hypomorphic Allele ◽  
Genes Encoding ◽  
Specific Proteins ◽  
Compound Heterozygous Mutations

Abstract Primary cilia contain specific proteins to achieve their functions as cellular antennae. Ciliary protein trafficking is mediated by the intraflagellar transport (IFT) machinery containing the IFT-A and IFT-B complexes. Mutations in genes encoding the IFT-A subunits (IFT43, IFT121/WDR35, IFT122, IFT139/TTC21B, IFT140, and IFT144/WDR19) often result in skeletal ciliopathies, including cranioectodermal dysplasia (CED). We here characterized the molecular and cellular defects of CED caused by compound heterozygous mutations in IFT144 [the missense variant IFT144(L710S) and the nonsense variant IFT144(R1103*)]. These two variants were distinct with regard to their interactions with other IFT-A subunits and with the IFT-B complex. When exogenously expressed in IFT144-knockout (KO) cells, IFT144(L710S) as well as IFT144(WT) rescued both moderately compromised ciliogenesis and the abnormal localization of ciliary proteins. As the homozygous IFT144(L710S) mutation was found to cause autosomal recessive retinitis pigmentosa, IFT144(L710S) is likely to be hypomorphic at the cellular level. In striking contrast, the exogenous expression of IFT144(R1103*) in IFT144-KO cells exacerbated the ciliogenesis defects. The expression of IFT144(R1103*) together with IFT144(WT) restored the abnormal phenotypes of IFT144-KO cells. However, the coexpression of IFT144(R1103*) with the hypomorphic IFT144(L710S) variant in IFT144-KO cells, which mimics the genotype of compound heterozygous CED patients, resulted in severe ciliogenesis defects. Taken together, these observations demonstrate that compound heterozygous mutations in IFT144 cause severe ciliary defects via a complicated mechanism, where one allele can cause severe ciliary defects when combined with a hypomorphic allele.

scholarly journals Selective Accumulation of Raft-Associated Membrane Protein Lat in T Cell Receptor Signaling Assemblies

2000 ◽  
Vol 151 (2) ◽  
pp. 199-208 ◽  
Author(s):  
Thomas Harder ◽  
Marina Kuhn
Keyword(s):  
Signal Transduction ◽  
Plasma Membrane ◽  
T Cell ◽  
Membrane Protein ◽  
Transmembrane Protein ◽  
Leukemic Cells ◽  
Signaling Proteins ◽  
Dependent Manner ◽  
Tcr Signal Transduction ◽  
Signal Transduction Proteins

Activation of T cell antigen receptor (TCR) induces tyrosine phosphorylations that mediate the assembly of signaling protein complexes. Moreover, cholesterol-sphingolipid raft membrane domains have been implicated to play a role in TCR signal transduction. Here, we studied the assembly of TCR with signal transduction proteins and raft markers in plasma membrane subdomains of Jurkat T leukemic cells. We employed a novel method to immunoisolate plasma membrane subfragments that were highly concentrated in activated TCR–CD3 complexes and associated signaling proteins. We found that the raft transmembrane protein linker for activation of T cells (LAT), but not a palmitoylation-deficient non-raft LAT mutant, strongly accumulated in TCR-enriched immunoisolates in a tyrosine phosphorylation–dependent manner. In contrast, other raft-associated molecules, including protein tyrosine kinases Lck and Fyn, GM1, and cholesterol, were not highly concentrated in TCR-enriched plasma membrane immunoisolates. Many downstream signaling proteins coisolated with the TCR/LAT-enriched plasma membrane fragments, suggesting that LAT/TCR assemblies form a structural scaffold for TCR signal transduction proteins. Our results indicate that TCR signaling assemblies in plasma membrane subdomains, rather than generally concentrating raft-associated membrane proteins and lipids, form by a selective protein-mediated anchoring of the raft membrane protein LAT in vicinity of TCR.

scholarly journals The initial events in myelin synthesis: orientation of proteolipid protein in the plasma membrane of cultured oligodendrocytes.

1989 ◽  
Vol 109 (2) ◽  
pp. 717-727 ◽  
Author(s):  
L D Hudson ◽  
V L Friedrich ◽  
T Behar ◽  
M Dubois-Dalcq ◽  
R A Lazzarini
Keyword(s):  
Plasma Membrane ◽  
Transmembrane Protein ◽  
Great Majority ◽  
Proteolipid Protein ◽  
The Other ◽  
Primary Sequence ◽  
Permeabilized Cells ◽  
Specific Proteins ◽  
The Central Nervous System ◽  
Peptide Antibodies

Proteolipid protein (PLP) is the most abundant transmembrane protein in myelin of the central nervous system. Conflicting models of PLP topology have been generated by computer predictions based on its primary sequence and experiments with purified myelin. We have examined the initial events in myelin synthesis, including the insertion and orientation of PLP in the plasma membrane, in rat oligodendrocytes which express PLP and the other myelin-specific proteins when cultured without neurons (Dubois-Dalcq, M., T. Behar, L. Hudson, and R. A. Lazzarini. 1986. J. Cell Biol. 102:384-392). These cells, identified by the presence of surface galactocerebroside, the major myelin glycolipid, were stained with six anti-peptide antibodies directed against hydrophilic or short hydrophobic sequences of PLP. Five of these anti-peptide antibodies specifically stained living oligodendrocytes. Staining was only seen approximately 10 d after PLP was first detected in the cytoplasm of fixed and permeabilized cells, suggesting that PLP is slowly transported from the RER to the cell surface. The presence of PLP domains on the extracellular surface was also confirmed by cleavage of such domains with proteases and by antibody-dependent complement-mediated lysis of living oligodendrocytes. Our results indicate that PLP has only two transmembrane domains and that the great majority of the protein, including its amino and carboxy termini, is located on the extracellular face of the oligodendrocyte plasma membrane. This disposition of the PLP molecule suggests that homophilic interactions between PLP molecules of apposed extracellular faces may mediate compaction of adjacent bilayers in the myelin sheath.

scholarly journals Uni-directional ciliary membrane protein trafficking by a cytoplasmic retrograde IFT motor and ciliary ectosome shedding

eLife ◽  
2015 ◽  
Vol 4 ◽  
Author(s):  
Muqing Cao ◽  
Jue Ning ◽  
Carmen I Hernandez-Lara ◽  
Olivier Belzile ◽  
Qian Wang ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Membrane Protein ◽  
Protein Trafficking ◽  
Protein Composition ◽  
Cytoplasmic Dynein ◽  
Cytoplasmic Microtubule ◽  
Dynamic Regulation ◽  
Ciliary Membrane ◽  
Membrane Protein Trafficking

The role of the primary cilium in key signaling pathways depends on dynamic regulation of ciliary membrane protein composition, yet we know little about the motors or membrane events that regulate ciliary membrane protein trafficking in existing organelles. Recently, we showed that cilium-generated signaling in Chlamydomonas induced rapid, anterograde IFT-independent, cytoplasmic microtubule-dependent redistribution of the membrane polypeptide, SAG1-C65, from the plasma membrane to the periciliary region and the ciliary membrane. Here, we report that the retrograde IFT motor, cytoplasmic dynein 1b, is required in the cytoplasm for this rapid redistribution. Furthermore, signaling-induced trafficking of SAG1-C65 into cilia is unidirectional and the entire complement of cellular SAG1-C65 is shed during signaling and can be recovered in the form of ciliary ectosomes that retain signal-inducing activity. Thus, during signaling, cells regulate ciliary membrane protein composition through cytoplasmic action of the retrograde IFT motor and shedding of ciliary ectosomes.

scholarly journals Plasma membrane domains enriched in cortical endoplasmic reticulum function as membrane protein trafficking hubs

2013 ◽  
Vol 24 (17) ◽  
pp. 2703-2713 ◽  
Author(s):  
Philip D. Fox ◽  
Christopher J. Haberkorn ◽  
Aubrey V. Weigel ◽  
Jenny L. Higgins ◽  
Elizabeth J. Akin ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Plasma Membrane ◽  
Membrane Protein ◽  
Membrane Trafficking ◽  
Protein Trafficking ◽  
Mammalian Cells ◽  
Transmembrane Protein ◽  
Surface Proteins ◽  
Hek 293 ◽  
Cortical Endoplasmic Reticulum

In mammalian cells, the cortical endoplasmic reticulum (cER) is a network of tubules and cisterns that lie in close apposition to the plasma membrane (PM). We provide evidence that PM domains enriched in underlying cER function as trafficking hubs for insertion and removal of PM proteins in HEK 293 cells. By simultaneously visualizing cER and various transmembrane protein cargoes with total internal reflectance fluorescence microscopy, we demonstrate that the majority of exocytotic delivery events for a recycled membrane protein or for a membrane protein being delivered to the PM for the first time occur at regions enriched in cER. Likewise, we observed recurring clathrin clusters and functional endocytosis of PM proteins preferentially at the cER-enriched regions. Thus the cER network serves to organize the molecular machinery for both insertion and removal of cell surface proteins, highlighting a novel role for these unique cellular microdomains in membrane trafficking.

scholarly journals Cholesterol accessibility at the ciliary membrane controls hedgehog signaling

eLife ◽  
2019 ◽  
Vol 8 ◽  
Author(s):  
Maia Kinnebrew ◽  
Ellen J Iverson ◽  
Bhaven B Patel ◽  
Ganesh V Pusapati ◽  
Jennifer H Kong ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Birth Defects ◽  
Hedgehog Signaling ◽  
Signal Transmission ◽  
Membrane Cholesterol ◽  
Signaling Proteins ◽  
Signaling System ◽  
Ciliary Membrane ◽  
Patched 1 ◽  
Hedgehog Signal

Previously we proposed that transmission of the hedgehog signal across the plasma membrane by Smoothened is triggered by its interaction with cholesterol (Luchetti et al., 2016). But how is cholesterol, an abundant lipid, regulated tightly enough to control a signaling system that can cause birth defects and cancer? Using toxin-based sensors that distinguish between distinct pools of cholesterol, we find that Smoothened activation and hedgehog signaling are driven by a biochemically-defined, small fraction of membrane cholesterol, termed accessible cholesterol. Increasing cholesterol accessibility by depletion of sphingomyelin, which sequesters cholesterol in complexes, amplifies hedgehog signaling. Hedgehog ligands increase cholesterol accessibility in the membrane of the primary cilium by inactivating the transporter-like protein Patched 1. Trapping this accessible cholesterol blocks hedgehog signal transmission across the membrane. Our work shows that the organization of cholesterol in the ciliary membrane can be modified by extracellular ligands to control the activity of cilia-localized signaling proteins.

scholarly journals Primary cilia and lipid raft dynamics

Open Biology ◽  
2021 ◽  
Vol 11 (8) ◽  
pp. 210130
Author(s):  
Yuhei Nishimura ◽  
Daishi Yamakawa ◽  
Katsunori Uchida ◽  
Takashi Shiromizu ◽  
Masatoshi Watanabe ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Lipid Rafts ◽  
Lipid Raft ◽  
Cancer Biology ◽  
Primary Cilia ◽  
Membrane Microdomains ◽  
Wide Range ◽  
Range Of Functions ◽  
Specific Proteins ◽  
The Relationship

Primary cilia, antenna-like structures of the plasma membrane, detect various extracellular cues and transduce signals into the cell to regulate a wide range of functions. Lipid rafts, plasma membrane microdomains enriched in cholesterol, sphingolipids and specific proteins, are also signalling hubs involved in a myriad of physiological functions. Although impairment of primary cilia and lipid rafts is associated with various diseases, the relationship between primary cilia and lipid rafts is poorly understood. Here, we review a newly discovered interaction between primary cilia and lipid raft dynamics that occurs during Akt signalling in adipogenesis. We also discuss the relationship between primary cilia and lipid raft-mediated Akt signalling in cancer biology. This review provides a novel perspective on primary cilia in the regulation of lipid raft dynamics.

scholarly journals Twin-arginine-dependent translocation of folded proteins

2012 ◽  
Vol 367 (1592) ◽  
pp. 1029-1046 ◽  
Author(s):  
Julia Fröbel ◽  
Patrick Rose ◽  
Matthias Müller
Keyword(s):  
Protein Transport ◽  
Signal Sequence ◽  
Transmembrane Protein ◽  
Proton Motive Force ◽  
Signal Peptides ◽  
Transport Pathway ◽  
Twin Arginine Translocation ◽  
Precursor Proteins ◽  
Binding Pockets ◽  
Folded Proteins

Twin-arginine translocation (Tat) denotes a protein transport pathway in bacteria, archaea and plant chloroplasts, which is specific for precursor proteins harbouring a characteristic twin-arginine pair in their signal sequences. Many Tat substrates receive cofactors and fold prior to translocation. For a subset of them, proofreading chaperones coordinate maturation and membrane-targeting. Tat translocases comprise two kinds of membrane proteins, a hexahelical TatC-type protein and one or two members of the single-spanning TatA protein family, called TatA and TatB. TatC- and TatA-type proteins form homo- and hetero-oligomeric complexes. The subunits of TatABC translocases are predominantly recovered from two separate complexes, a TatBC complex that might contain some TatA, and a homomeric TatA complex. TatB and TatC coordinately recognize twin-arginine signal peptides and accommodate them in membrane-embedded binding pockets. Advanced binding of the signal sequence to the Tat translocase requires the proton-motive force (PMF) across the membranes and might involve a first recruitment of TatA. When targeted in this manner, folded twin-arginine precursors induce homo-oligomerization of TatB and TatA. Ultimately, this leads to the formation of a transmembrane protein conduit that possibly consists of a pore-like TatA structure. The translocation step again is dependent on the PMF.

scholarly journals The ciliary targeting of membrane proteins by a ternary complex comprising transportin1, Rab8 and the ciliary targeting signal

2016 ◽  
Author(s):  
Viswanadh Madugula ◽  
Lei Lu
Keyword(s):  
Ternary Complex ◽  
Molecular Level ◽  
Nucleotide Exchange ◽  
Lateral Transport ◽  
Retinol Dehydrogenase ◽  
Transport Pathway ◽  
Binding Motifs ◽  
Ciliary Membrane ◽  
Guanine Nucleotide Exchange ◽  
Targeting Signal

AbstractThe sensory functions of cilia are dependent on the enrichment of ciliary resident proteins. While it is known that ciliary targeting signals (CTSs) specifically target ciliary proteins to cilia, it is still unclear how CTSs facilitate the entry and retention of ciliary residents at the molecular level. We found that non-ciliary membrane reporters can passively diffuse to cilia via the lateral transport pathway and the translocation of membrane reporters through the ciliary diffusion barrier is facilitated by importin binding motifs/domains. Screening known CTSs of ciliary membrane residents uncovered that fibrocystin, photoreceptor retinol dehydrogenase, rhodopsin and retinitis pigmentosa 2 interact with transportin1 (TNPO1) via previously identified CTSs. We further discovered that a novel ternary complex, comprising TNPO1, Rab8 and CTS, can assemble or disassemble under the guanine nucleotide exchange of Rab8. Our study suggests a novel mechanism in which TNPO1/Rab8/CTS complex mediates selective entry and retention of cargos within cilia.

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