An organelle-specific protein landscape identifies novel diseases and molecular mechanisms

Abstract

Cellular organelles provide opportunities to relate biological mechanisms to disease. Here we use affinity proteomics, genetics and cell biology to interrogate cilia: poorly understood organelles, where defects cause genetic diseases. Two hundred and seventeen tagged human ciliary proteins create a final landscape of 1,319 proteins, 4,905 interactions and 52 complexes. Reverse tagging, repetition of purifications and statistical analyses, produce a high-resolution network that reveals organelle-specific interactions and complexes not apparent in larger studies, and links vesicle transport, the cytoskeleton, signalling and ubiquitination to ciliary signalling and proteostasis. We observe sub-complexes in exocyst and intraflagellar transport complexes, which we validate biochemically, and by probing structurally predicted, disruptive, genetic variants from ciliary disease patients. The landscape suggests other genetic diseases could be ciliary including 3M syndrome. We show that 3M genes are involved in ciliogenesis, and that patient fibroblasts lack cilia. Overall, this organelle-specific targeting strategy shows considerable promise for Systems Medicine.

The Intraflagellar Transport Machinery

“…2). This conclusion, based on the analysis of purified Chlamydomonas proteins, was recently confirmed using a "visible immunoprecipitation" (VIP) approach with human proteins (Katoh et al 2016), as well as in human cells in a large-scale proteomic study (Boldt et al 2016). With regard to salt stability, it should be noted that five of the six IFT-B2 proteins are stable at NaCl concentrations of 1 M (Taschner et al 2016), with IFT172 being the only weakly attached subunit, confirming previously published data (Cole et al 1998;Pedersen et al 2005).…”

“…A similar destabilization of IFT-B1 (IFT81 and IFT46) and IFT-B2 (IFT57 and IFT20) proteins as well as a slight increase in an IFT-A protein (IFT139) were recently observed in an IFT74 (ift74-2) Chlamydomonas mutant (Brown et al 2015). IFT88 is not required for the stability of the IFT-B1 complex (Pazour et al 2000;Richey and Qin 2012;Taschner et al 2014), but as indicated above is part of the interface between IFT-B1 and IFT-B2 (Boldt et al 2016;Taschner et al 2016). Consistently, the IFT-B complex formed in the ift88 mutant is smaller (Richey and Qin 2012), and IFT57 (IFT-B2 component) but not IFT81 (IFT-B1 component) is destabilized (Pazour et al 2000).…”

“…Although some studies were unable to detect coprecipiation of IFT-A and IFT-B subunits (for example, see Cole et al 1998;Baker et al 2003;Follit et al 2009;Lechtreck et al 2009b;Fan et al 2010), other studies did detect an interaction (Qin et al 2004;Pedersen et al 2005), although coprecipitation seemed to be dependent on the antibodies and pulldown conditions used. High-throughput pulldowns and mass-spectrometry analysis did not detect direct interactions between IFT-A and IFT-B (Boldt et al 2016), but a recent study using Chlamydomonas provided evidence that the amino-terminal part of the IFT-B1 component IFT74 could be linking IFT-B and IFT-A. In a mutant strain missing this IFT74 region, the IFT-A complex was unable to enter the flagellum, and IFT-B components accumulated at a tip as a result (Brown et al 2015).…”

Regulation of ciliary retrograde protein trafficking by the Joubert syndrome proteins ARL13B and INPP5E

“…Using our VIP assay, we previously determined the overall architectures of the exocyst, BBSome and IFT-B complexes. The IFT-B complex is composed of 16 subunits that can be divided into the core and peripheral subcomplexes, composed of 10 subunits [IFT22, IFT25 (also known as HSPB11), IFT27, IFT46, IFT52, IFT56, IFT70 (also known as TTC30B), IFT74, IFT81 and IFT88] (Lucker et al, 2005;Taschner et al, 2014) and 6 subunits [IFT20, IFT38 (also known as CLUAP1), IFT54 (also known as TRAF3IP1), IFT57, IFT80 and IFT172], respectively, which are connected to each other by composite interactions involving IFT38, IFT52, IFT57 and IFT88 (Boldt et al, 2016;Katoh et al, 2016;Taschner et al, 2016) (also see Fig. 8).…”

Identification of Protein Complexes Associated with the Usher Syndrome 2C and Epilepsy-Associated Protein VLGR1 Applying Affinity Proteomics

“…For this, we applied high-resolution affinity proteomics based on tandem affinity purifications (TAPs). We systematically tagged domains of VLGR1 and full-length VLGR1a with the Strep II Flag (SF)-tag ( Figure 1A) and expressed these baits in HEK293T cells for TAPs of protein complexes related to VLGR1 [9,10]. Subsequently, we determined the composition of eluted protein complexes by mass spectrometry.…”