scholarly journals Structure of the human BBSome core complex in the open conformation

2019 ◽  
Author(s):  
Björn U. Klink ◽  
Christos Gatsogiannis ◽  
Oliver Hofnagel ◽  
Alfred Wittinghofer ◽  
Stefan Raunser
Keyword(s):  
High Resolution ◽  
Primary Cilia ◽  
Intraflagellar Transport ◽  
Underlying Mechanism ◽  
Signaling Proteins ◽  
Core Complex ◽  
Bardet Biedl Syndrome ◽  
Open Conformation ◽  
Cargo Proteins ◽  
Positively Charged

AbstractThe BBSome is a heterooctameric protein complex that plays a central role in primary cilia homeostasis. Its malfunction causes the severe ciliopathy Bardet-Biedl syndrome (BBS). The complex acts as a cargo adapter that recognizes signaling proteins such as GPCRs and links them to the intraflagellar transport machinery. The underlying mechanism is poorly understood. Here we present a high-resolution cryo-EM structure of a human heterohexameric core subcomplex of the BBSome. The structure reveals the architecture of the complex in atomic detail. It explains how the subunits interact with each other and how disease-causing mutations hamper this interaction. The complex adopts a conformation that is open for binding to membrane-associated GTPase Arl6 and a large positively charged patch likely strengthens the interaction with the membrane. A prominent negatively charged cleft at the center of the complex is likely involved in binding of positively charged signaling sequences of cargo proteins.

scholarly journals Structure of the human BBSome core complex

eLife ◽  
2020 ◽  
Vol 9 ◽  
Author(s):  
Björn Udo Klink ◽  
Christos Gatsogiannis ◽  
Oliver Hofnagel ◽  
Alfred Wittinghofer ◽  
Stefan Raunser
Keyword(s):  
High Resolution ◽  
Protein Complex ◽  
Primary Cilia ◽  
Intraflagellar Transport ◽  
Underlying Mechanism ◽  
Signaling Proteins ◽  
Core Complex ◽  
Bardet Biedl Syndrome ◽  
Cargo Proteins ◽  
Positively Charged

The BBSome is a heterooctameric protein complex that plays a central role in primary cilia homeostasis. Its malfunction causes the severe ciliopathy Bardet-Biedl syndrome (BBS). The complex acts as a cargo adapter that recognizes signaling proteins such as GPCRs and links them to the intraflagellar transport machinery. The underlying mechanism is poorly understood. Here we present a high-resolution cryo-EM structure of a human heterohexameric core subcomplex of the BBSome. The structure reveals the architecture of the complex in atomic detail. It explains how the subunits interact with each other and how disease-causing mutations hamper this interaction. The complex adopts a conformation that is open for binding to membrane-associated GTPase Arl6 and a large positively charged patch likely strengthens the interaction with the membrane. A prominent negatively charged cleft at the center of the complex is likely involved in binding of positively charged signaling sequences of cargo proteins.

scholarly journals Intraflagellar transport protein RABL5/IFT22 recruits the BBSome to the basal body through the GTPase ARL6/BBS3

2020 ◽  
Vol 117 (5) ◽  
pp. 2496-2505 ◽  
Author(s):  
Bin Xue ◽  
Yan-Xia Liu ◽  
Bin Dong ◽  
Jenna L. Wingfield ◽  
Mingfu Wu ◽  
...  
Keyword(s):  
Basal Body ◽  
In Vivo Imaging ◽  
Bound States ◽  
Intraflagellar Transport ◽  
Signaling Proteins ◽  
Gtpase Activity ◽  
Guanosine Triphosphate ◽  
Bardet Biedl Syndrome ◽  
Intrinsic Gtpase Activity

Bardet-Biedl syndrome (BBS) is a ciliopathy caused by defects in the assembly or distribution of the BBSome, a conserved protein complex. The BBSome cycles via intraflagellar transport (IFT) through cilia to transport signaling proteins. How the BBSome is recruited to the basal body for binding to IFT trains for ciliary entry remains unknown. Here, we show that the Rab-like 5 GTPase IFT22 regulates basal body targeting of the BBSome in Chlamydomonas reinhardtii. Our functional, biochemical and single particle in vivo imaging assays show that IFT22 is an active GTPase with low intrinsic GTPase activity. IFT22 is part of the IFT-B1 subcomplex but is not required for ciliary assembly. Independent of its association to IFT-B1, IFT22 binds and stabilizes the Arf-like 6 GTPase BBS3, a BBS protein that is not part of the BBSome. IFT22/BBS3 associates with the BBSome through an interaction between BBS3 and the BBSome. When both IFT22 and BBS3 are in their guanosine triphosphate (GTP)-bound states they recruit the BBSome to the basal body for coupling with the IFT-B1 subcomplex. The GTP-bound BBS3 likely remains to be associated with the BBSome upon ciliary entry. In contrast, IFT22 is not required for the transport of BBSomes in cilia, indicating that the BBSome is transferred from IFT22 to the IFT trains at the ciliary base. In summary, our data propose that nucleotide-dependent recruitment of the BBSome to the basal body by IFT22 regulates BBSome entry into cilia.

scholarly journals Bardet-Biedl Syndrome 3 protein promotes ciliary exit of the signaling protein phospholipase D via the BBSome

eLife ◽  
2021 ◽  
Vol 10 ◽  
Author(s):  
Yan-Xia Liu ◽  
Bin Xue ◽  
Wei-Yue Sun ◽  
Jenna L Wingfield ◽  
Jun Sun ◽  
...  
Keyword(s):  
Chlamydomonas Reinhardtii ◽  
Phospholipase D ◽  
Basal Body ◽  
Regulatory Mechanism ◽  
Intraflagellar Transport ◽  
Bound State ◽  
Signaling Proteins ◽  
Bardet Biedl Syndrome ◽  
Signaling Protein ◽  
Ciliary Signaling

Certain ciliary signaling proteins couple with the BBSome, a conserved complex of Bardet-Biedl syndrome (BBS) proteins, to load onto retrograde intraflagellar transport (IFT) trains for their removal out of cilia in Chlamydomonas reinhardtii. Here, we show that loss of the Arf-like 6 (ARL6) GTPase BBS3 causes the signaling protein phospholipase D (PLD) to accumulate in cilia. Upon targeting to the basal body, BBSomes enter and cycle through cilia via IFT, while BBS3 in a GTP-bound state separates from BBSomes, associates with the membrane, and translocates from the basal body to cilia by diffusion. Upon arriving at the ciliary tip, GTP-bound BBS3 binds and recruits BBSomes to the ciliary membrane for interacting with PLD, thus making the PLD-laden BBSomes available to load onto retrograde IFT trains for ciliary exit. Therefore, BBS3 promotes PLD exit from cilia via the BBSome providing a regulatory mechanism for ciliary signaling protein removal out of cilia.

scholarly journals The Chlamydomonas reinhardtii BBSome is an IFT cargo required for export of specific signaling proteins from flagella

2009 ◽  
Vol 187 (7) ◽  
pp. 1117-1132 ◽  
Author(s):  
Karl-Ferdinand Lechtreck ◽  
Eric C. Johnson ◽  
Tsuyoshi Sakai ◽  
Deborah Cochran ◽  
Bryan A. Ballif ◽  
...  
Keyword(s):  
Chlamydomonas Reinhardtii ◽  
Intraflagellar Transport ◽  
Full Length ◽  
Signaling Proteins ◽  
Related Disorder ◽  
Bardet Biedl Syndrome ◽  
Insertional Mutants ◽  
Integral Component ◽  
Evolutionarily Conserved ◽  
Conserved Genes

In humans, seven evolutionarily conserved genes that cause the cilia-related disorder Bardet-Biedl syndrome (BBS) encode proteins that form a complex termed the BBSome. The function of the BBSome in the cilium is not well understood. We purified a BBSome-like complex from Chlamydomonas reinhardtii flagella and found that it contains at least BBS1, -4, -5, -7, and -8 and undergoes intraflagellar transport (IFT) in association with a subset of IFT particles. C. reinhardtii insertional mutants defective in BBS1, -4, and -7 assemble motile, full-length flagella but lack the ability to phototax. In the bbs4 mutant, the assembly and transport of IFT particles are unaffected, but the flagella abnormally accumulate several signaling proteins that may disrupt phototaxis. We conclude that the BBSome is carried by IFT but is an adapter rather than an integral component of the IFT machinery. C. reinhardtii BBS4 may be required for the export of signaling proteins from the flagellum via IFT.

scholarly journals ERICH3 in Primary Cilia Regulates Cilium Formation and the Localisations of Ciliary Transport and Sonic Hedgehog Signaling Proteins

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Mona Alsolami ◽  
Stefanie Kuhns ◽  
Manal Alsulami ◽  
Oliver E. Blacque
Keyword(s):  
Sonic Hedgehog ◽  
Primary Cilium ◽  
Molecular Motors ◽  
Hedgehog Signaling ◽  
Primary Cilia ◽  
Intraflagellar Transport ◽  
Retrograde Transport ◽  
Signaling Proteins ◽  
Sonic Hedgehog Signaling ◽  
And Function

Abstract Intraflagellar transport (IFT) is essential for the formation and function of the microtubule-based primary cilium, which acts as a sensory and signalling device at the cell surface. Consisting of IFT-A/B and BBSome cargo adaptors that associate with molecular motors, IFT transports protein into (anterograde IFT) and out of (retrograde IFT) the cilium. In this study, we identify the mostly uncharacterised ERICH3 protein as a component of the mammalian primary cilium. Loss of ERICH3 causes abnormally short cilia and results in the accumulation of IFT-A/B proteins at the ciliary tip, together with reduced ciliary levels of retrograde transport regulators, ARL13B, INPP5E and BBS5. We also show that ERICH3 ciliary localisations require ARL13B and BBSome components. Finally, ERICH3 loss causes positive (Smoothened) and negative (GPR161) regulators of sonic hedgehog signaling (Shh) to accumulate at abnormally high levels in the cilia of pathway-stimulated cells. Together, these findings identify ERICH3 as a novel component of the primary cilium that regulates cilium length and the ciliary levels of Shh signaling molecules. We propose that ERICH3 functions within retrograde IFT-associated pathways to remove signaling proteins from cilia.

scholarly journals Chlamydomonas LZTFL1 mediates phototaxis via controlling BBSome recruitment to the basal body and its reassembly at the ciliary tip

2021 ◽  
Vol 118 (35) ◽  
pp. e2101590118
Author(s):  
Wei-Yue Sun ◽  
Bin Xue ◽  
Yan-Xia Liu ◽  
Rui-Kai Zhang ◽  
Rong-Chao Li ◽  
...  
Keyword(s):  
Basal Body ◽  
Leucine Zipper ◽  
Intraflagellar Transport ◽  
G Protein Coupled Receptors ◽  
Signaling Proteins ◽  
Dual Mode ◽  
Bardet Biedl Syndrome ◽  
Cellular Processes ◽  
G Protein Coupled ◽  
Ciliary Signaling

Many G protein–coupled receptors and other signaling proteins localize to the ciliary membrane for regulating diverse cellular processes. The BBSome composed of multiple Bardet–Biedl syndrome (BBS) proteins is an intraflagellar transport (IFT) cargo adaptor essential for sorting signaling proteins in and/or out of cilia via IFT. Leucine zipper transcription factor-like 1 (LZTFL1) protein mediates ciliary signaling by controlling BBSome ciliary content, reflecting how LZTFL1 mutations could cause BBS. However, the mechanistic mechanism underlying this process remains elusive thus far. Here, we show that LZTFL1 maintains BBSome ciliary dynamics by finely controlling BBSome recruitment to the basal body and its reassembly at the ciliary tip simultaneously in Chlamydomonas reinhardtii. LZTFL1 directs BBSome recruitment to the basal body via promoting basal body targeting of Arf-like 6 GTPase BBS3, thus deciding the BBSome amount available for loading onto anterograde IFT trains for entering cilia. Meanwhile, LZTFL1 stabilizes the IFT25/27 component of the IFT-B1 subcomplex in the cell body so as to control its presence and amount at the basal body for entering cilia. Since IFT25/27 promotes BBSome reassembly at the ciliary tip for loading onto retrograde IFT trains, LZTFL1 thus also directs BBSome removal out of cilia. Therefore, LZTFL1 dysfunction deprives the BBSome of ciliary presence and generates Chlamydomonas cells defective in phototaxis. In summary, our data propose that LZTFL1 maintains BBSome dynamics in cilia by such a dual-mode system, providing insights into how LZTFL1 mediates ciliary signaling through maintaining BBSome ciliary dynamics and the pathogenetic mechanism of the BBS disorder as well.

scholarly journals SNX27-FERM-SNX1 complex structure rationalizes divergent trafficking pathways by SNX17 and SNX27

2021 ◽  
Vol 118 (36) ◽  
pp. e2105510118
Author(s):  
Xin Yong ◽  
Lin Zhao ◽  
Wenfeng Hu ◽  
Qingxiang Sun ◽  
Hyoungjun Ham ◽  
...  
Keyword(s):  
Complex Structure ◽  
Pdz Domain ◽  
Underlying Mechanism ◽  
Endocytic Recycling ◽  
Ferm Domain ◽  
Binding Motifs ◽  
Molecular Events ◽  
N Terminus ◽  
Cargo Proteins ◽  
Positively Charged

The molecular events that determine the recycling versus degradation fates of internalized membrane proteins remain poorly understood. Two of the three members of the SNX-FERM family, SNX17 and SNX31, utilize their FERM domain to mediate endocytic trafficking of cargo proteins harboring the NPxY/NxxY motif. In contrast, SNX27 does not recycle NPxY/NxxY-containing cargo but instead recycles cargo containing PDZ-binding motifs via its PDZ domain. The underlying mechanism governing this divergence in FERM domain binding is poorly understood. Here, we report that the FERM domain of SNX27 is functionally distinct from SNX17 and interacts with a novel DLF motif localized within the N terminus of SNX1/2 instead of the NPxY/NxxY motif in cargo proteins. The SNX27-FERM-SNX1 complex structure reveals that the DLF motif of SNX1 binds to a hydrophobic cave surrounded by positively charged residues on the surface of SNX27. The interaction between SNX27 and SNX1/2 is critical for efficient SNX27 recruitment to endosomes and endocytic recycling of multiple cargoes. Finally, we show that the interaction between SNX27 and SNX1/2 is critical for brain development in zebrafish. Altogether, our study solves a long-standing puzzle in the field and suggests that SNX27 and SNX17 mediate endocytic recycling through fundamentally distinct mechanisms.

scholarly journals Intraflagellar transport molecules in ciliary and nonciliary cells of the retina

2010 ◽  
Vol 189 (1) ◽  
pp. 171-186 ◽  
Author(s):  
Tina Sedmak ◽  
Uwe Wolfrum
Keyword(s):  
Molecular Motors ◽  
Primary Cilia ◽  
Intraflagellar Transport ◽  
Retinal Neurons ◽  
Core Complex ◽  
Cytoplasmic Transport ◽  
Transport Vesicles ◽  
Transport Molecules ◽  
Established Role ◽  
Intense Research

The assembly and maintenance of cilia require intraflagellar transport (IFT), a process mediated by molecular motors and IFT particles. Although IFT is a focus of current intense research, the spatial distribution of individual IFT proteins remains elusive. In this study, we analyzed the subcellular localization of IFT proteins in retinal cells by high resolution immunofluorescence and immunoelectron microscopy. We report that IFT proteins are differentially localized in subcompartments of photoreceptor cilia and in defined periciliary target domains for cytoplasmic transport, where they are associated with transport vesicles. IFT20 is not in the IFT core complex in photoreceptor cilia but accompanies Golgi-based sorting and vesicle trafficking of ciliary cargo. Moreover, we identify a nonciliary IFT system containing a subset of IFT proteins in dendrites of retinal neurons. Collectively, we provide evidence to implicate the differential composition of IFT systems in cells with and without primary cilia, thereby supporting new functions for IFT beyond its well-established role in cilia.

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.

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