High-Efficiency Isolation of Nuclear Envelope Protein Complexes from Trypanosomes

Obado, Samson O.; Field, Mark C.; Chait, Brian T.; Rout, Michael P.

doi:10.1007/978-1-4939-3530-7_3

Cited by 34 publications

(35 citation statements)

References 10 publications

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“…Cultures of each cell line were snap-frozen and subjected to cryomilling to generate a powder [ 55 ]. Aliquots of this powder were used to systematically optimise conditions for isolation of PABP complexes.…”

Section: Resultsmentioning

confidence: 99%

Comparative proteomics of the two T. brucei PABPs suggests that PABP2 controls bulk mRNA

et al. 2018

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Poly(A)-binding proteins (PABPs) regulate mRNA fate by controlling stability and translation through interactions with both the poly(A) tail and eIF4F complex. Many organisms have several paralogs of PABPs and eIF4F complex components and it is likely that different eIF4F/PABP complex combinations regulate distinct sets of mRNAs. Trypanosomes have five eIF4G paralogs, six of eIF4E and two PABPs, PABP1 and PABP2. Under starvation, polysomes dissociate and the majority of mRNAs, most translation initiation factors and PABP2 reversibly localise to starvation stress granules. To understand this more broadly we identified a protein interaction cohort for both T. brucei PABPs by cryo-mill/affinity purification-mass spectrometry. PABP1 very specifically interacts with the previously identified interactors eIF4E4 and eIF4G3 and few others. In contrast PABP2 is promiscuous, with a larger set of interactors including most translation initiation factors and most prominently eIF4G1, with its two partners TbG1-IP and TbG1-IP2. Only RBP23 was specific to PABP1, whilst 14 RNA-binding proteins were exclusively immunoprecipitated with PABP2. Significantly, PABP1 and associated proteins are largely excluded from starvation stress granules, but PABP2 and most interactors translocate to granules on starvation. We suggest that PABP1 regulates a small subpopulation of mainly small-sized mRNAs, as it interacts with a small and distinct set of proteins unable to enter the dominant pathway into starvation stress granules and localises preferentially to a subfraction of small polysomes. By contrast PABP2 likely regulates bulk mRNA translation, as it interacts with a wide range of proteins, enters stress granules and distributes over the full range of polysomes.

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Section: Resultsmentioning

confidence: 99%

Comparative proteomics of the two T. brucei PABPs suggests that PABP2 controls bulk mRNA

et al. 2018

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“…brucei by affinity isolation and cryomilling (see methods) using a genomically-tagged TbSec15::GFP fusion protein as an affinity handle [42]. Initially, immunoprecipitations (IPs) were performed using various buffers and analysed by SDS-PAGE.…”

Section: Resultsmentioning

confidence: 99%

The Trypanosome Exocyst: A Conserved Structure Revealing a New Role in Endocytosis

et al. 2017

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Membrane transport is an essential component of pathogenesis for most infectious organisms. In African trypanosomes, transport to and from the plasma membrane is closely coupled to immune evasion and antigenic variation. In mammals and fungi an octameric exocyst complex mediates late steps in exocytosis, but comparative genomics suggested that trypanosomes retain only six canonical subunits, implying mechanistic divergence. We directly determined the composition of the Trypanosoma brucei exocyst by affinity isolation and demonstrate that the parasite complex is nonameric, retaining all eight canonical subunits (albeit highly divergent at the sequence level) plus a novel essential subunit, Exo99. Exo99 and Sec15 knockdowns have remarkably similar phenotypes in terms of viability and impact on morphology and trafficking pathways. Significantly, both Sec15 and Exo99 have a clear function in endocytosis, and global proteomic analysis indicates an important role in maintaining the surface proteome. Taken together these data indicate additional exocyst functions in trypanosomes, which likely include endocytosis, recycling and control of surface composition. Knockdowns in HeLa cells suggest that the role in endocytosis is shared with metazoan cells. We conclude that, whilst the trypanosome exocyst has novel components, overall functionality appears conserved, and suggest that the unique subunit may provide therapeutic opportunities.

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“…To examine this possibility, we immunoisolated the Vps8a containing CORVET complex by affinity capture using Vps8a-FLAG as bait. To overcome any issues from low expression levels we adapted methods used in trypanosomes (Obado et al, 2016), yeast (Oeffinger et al, 2007) and mammalian cells (LaCava et al, 2016), where large numbers of cells are rapidly frozen and milled to generate cryopowders. We tested a variety of buffer conditions for solubilizing Vps8a from the cryopowders and final conditions resulted in ~60% solubilization (not shown).…”

Section: Vps8ap Defines a Specific Hexameric Corvet Complex In Tetrahmentioning

confidence: 99%

Diversification of CORVET tethers facilitates transport complexity inTetrahymena thermophila

Sparvoli

Zoltner

Field

et al. 2019

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In endolysosomal networks, two hetero-hexameric tethers called HOPS and CORVET are found widely throughout eukaryotes. The unicellular ciliate Tetrahymena thermophila possesses elaborate endolysosomal structures, but curiously both it and related protozoa lack the HOPS tether and several other trafficking genes while retaining the related CORVET complex. Tetrahymena encodes multiple paralogs of most CORVET subunits, which assemble into six distinct complexes. Each complex has a unique subunit composition and, significantly, shows unique localization, indicating participation in distinct pathways. One pair of complexes differ by a single subunit (Vps8), but have late endosomal vs. recycling endosome locations. While Vps8 subunits are thus prime determinants for targeting and functional specificity, determinants exist on all subunits except Vps11. This unprecedented expansion and diversification of CORVET provides a potent example of tether flexibility, and illustrates how 'backfilling' following secondary losses of trafficking genes can provide a mechanism for evolution of new pathways. 3

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High-Efficiency Isolation of Nuclear Envelope Protein Complexes from Trypanosomes

Cited by 34 publications

References 10 publications

Comparative proteomics of the two T. brucei PABPs suggests that PABP2 controls bulk mRNA

Comparative proteomics of the two T. brucei PABPs suggests that PABP2 controls bulk mRNA

The Trypanosome Exocyst: A Conserved Structure Revealing a New Role in Endocytosis

Diversification of CORVET tethers facilitates transport complexity inTetrahymena thermophila

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