BACKGROUND: Comparing a parasitic lineage to its free-living relatives is a powerful way to understand how that evolutionary transition to parasitism occurred. Giardia intestinalis (Fornicata) is a leading cause of gastrointestinal disease world-wide and is famous for its unusual complement of cellular compartments, such as having peripheral vacuoles instead of typical endosomal compartments. Endocytosis plays an important role in Giardia's pathogenesis. Endosomal sorting complexes required for transport (ESCRT) are membrane-deforming proteins associated with the late endosome/multivesicular body (MVB). MVBs are ill-defined in G. intestinalis, and roles for identified ESCRT-related proteins are not fully understood in the context of its unique endocytic system. Furthermore, components thought to be required for full ESCRT functionality have not yet been documented in this species. RESULTS: We used genomic and transcriptomic data from several Fornicata species to clarify the evolutionary genome streamlining observed in Giardia, as well as to detect any divergent orthologs of the Fornicata ESCRT subunits. We observed differences in the ESCRT machinery complement between Giardia strains. Microscopy-based investigations of key components of ESCRT machinery such as GiVPS36 and GiVPS25 link them to peripheral vacuoles, highlighting these organelles as simplified MVB equivalents. Unexpectedly, we show ESCRT components associated with the endoplasmic reticulum and, for the first time, mitosomes. Finally, we identified the rare ESCRT component CHMP7 in several fornicate representatives, including Giardia and show that contrary to current understanding, CHMP7 evolved from a gene fusion of VPS25 and SNF7 domains, prior to the last eukaryotic common ancestor, over 1.5 billion years ago. CONCLUSIONS: Our findings show that ESCRT machinery in G. intestinalis is far more varied and complete than previously thought, associates to multiple cellular locations, and presents changes in ESCRT complement which pre-date adoption of a parasitic lifestyle.
The factors and mechanisms involved in vacuolar transport in plants, and in particular those directing vesicles to their target endomembrane compartment, remain largely unknown. To identify components of the vacuolar trafficking machinery, we searched for Arabidopsis modified transport to the vacuole (mtv) mutants that abnormally secrete the synthetic vacuolar cargo VAC2. We report here on the identification of 17 mtv mutations, corresponding to mutant alleles of MTV2/VSR4, MTV3/PTEN2A MTV7/EREL1, MTV8/ARFC1, MTV9/PUF2, MTV10/VPS3, MTV11/VPS15, MTV12/GRV2, MTV14/GFS10, MTV15/BET11, MTV16/VPS51, MTV17/VPS54, and MTV18/VSR1 Eight of the MTV proteins localize at the interface between the trans-Golgi network (TGN) and the multivesicular bodies (MVBs), supporting that the trafficking step between these compartments is essential for segregating vacuolar proteins from those destined for secretion. Importantly, the GARP tethering complex subunits MTV16/VPS51 and MTV17/VPS54 were found at endoplasmic reticulum (ER)- and microtubule-associated compartments (EMACs). Moreover, MTV16/VPS51 interacts with the motor domain of kinesins, suggesting that, in addition to tethering vesicles, the GARP complex may regulate the motors that transport them. Our findings unveil a previously uncharacterized compartment of the plant vacuolar trafficking pathway and support a role for microtubules and kinesins in GARP-dependent transport of soluble vacuolar cargo in plants.
- MeSH
- alely MeSH
- Arabidopsis genetika metabolismus MeSH
- cytoplazmatické vezikuly genetika metabolismus MeSH
- endoplazmatické retikulum genetika metabolismus MeSH
- Golgiho aparát genetika metabolismus MeSH
- kineziny genetika metabolismus MeSH
- mikrotubuly genetika metabolismus MeSH
- multivezikulární tělíska genetika metabolismus MeSH
- mutace MeSH
- proteiny huseníčku genetika metabolismus MeSH
- transport proteinů genetika MeSH
- vakuoly genetika metabolismus MeSH
- vezikulární transportní proteiny genetika metabolismus MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
The Arabidopsisthaliana pathogenesis-related 1 (PR1) is an important defense protein, so far it has only been detected in extracellular space and its subcellular sorting and transport remain unexplained. Using a green fluorescent protein (GFP) tagged full length, as well as a C-terminus truncated version of PR1, we observed that when expressed ectopically in Nicotiana benthamiana leaves, PR1 co-localizes only partially with Golgi markers, and much more prominently with the late endosome (LE)/multivesicular body (MVB) FYVE marker. The C-truncated version PR1ΔC predominantly localized to the endoplasmic reticulum (ER). The same localizations were found for stable Arabidopsis transformants with expression of PR1 and PR1ΔC driven by the native promoter. We conclude that the A. thaliana PR1 (AtPR1) undergoes an unconventional secretion pathway, starting from the C-terminus-dependent sorting from the ER, and utilizing further transportation via phosphatidyl-inositol-3-phosphate (PI(3)P) positive LE/MVB-like vesicles. The homology model of the PR1 structure shows that the cluster of positively charged amino acid residues (arginines 60, 67, 137, and lysine 135) could indeed interact with negatively charged phospholipids of cellular membranes. It remains to be resolved whether Golgi and LE/MVB localization reflects an alternative sorting or trafficking succession, and what the role of lipid interactions in it will be.
- MeSH
- Arabidopsis metabolismus MeSH
- endoplazmatické retikulum metabolismus MeSH
- endozomy metabolismus MeSH
- fosfatidylinositolfosfáty metabolismus MeSH
- Golgiho aparát metabolismus MeSH
- konfokální mikroskopie MeSH
- listy rostlin metabolismus MeSH
- promotorové oblasti (genetika) MeSH
- proteiny huseníčku genetika metabolismus MeSH
- rekombinantní fúzní proteiny biosyntéza genetika MeSH
- tabák metabolismus MeSH
- zelené fluorescenční proteiny genetika metabolismus MeSH
- Publikační typ
- časopisecké články MeSH
Exosomes are small vesicles that are secreted by cells and act as mediators of cell to cell communication. Because of their potential therapeutic significance, important efforts are being made towards characterizing exosomal contents. However, little is known about the mechanisms that govern exosome biogenesis. We have recently shown that the exosomal protein syntenin supports exosome production. Here we identify the small GTPase ADP ribosylation factor 6 (ARF6) and its effector phospholipase D2 (PLD2) as regulators of syntenin exosomes. ARF6 and PLD2 affect exosomes by controlling the budding of intraluminal vesicles (ILVs) into multivesicular bodies (MVBs). ARF6 also controls epidermal growth factor receptor degradation, suggesting a role in degradative MVBs. Yet ARF6 does not affect HIV-1 budding, excluding general effects on Endosomal Sorting Complexes Required for Transport. Our study highlights a novel pathway controlling ILV budding and exosome biogenesis and identifies an unexpected role for ARF6 in late endosomal trafficking.
- MeSH
- ADP-ribosylační faktory genetika metabolismus MeSH
- buněčné linie MeSH
- endozomální třídící komplexy pro transport genetika metabolismus MeSH
- erbB receptory metabolismus MeSH
- exozómy enzymologie genetika metabolismus MeSH
- fosfolipasa D genetika metabolismus MeSH
- HIV infekce genetika metabolismus virologie MeSH
- HIV-1 fyziologie MeSH
- lidé MeSH
- multivezikulární tělíska enzymologie genetika metabolismus MeSH
- proteiny buněčného cyklu genetika metabolismus MeSH
- proteiny vázající vápník genetika metabolismus MeSH
- synteniny genetika metabolismus MeSH
- transport proteinů MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
Wortmannin is a widely used pharmaceutical compound which is employed to define vesicular trafficking routes of particular proteins or cellular compounds. It targets phosphatidylinositol 3-kinase and phosphatidylinositol 4-kinases in a dose-dependent manner leading to the inhibition of protein vacuolar sorting and endocytosis. Combined proteomics and cell biological approaches have been used in this study to explore the effects of wortmannin on Arabidopsis root cells, especially on proteome and endomembrane trafficking. On the subcellular level, wortmannin caused clustering, fusion, and swelling of trans-Golgi network (TGN) vesicles and multivesicular bodies (MVBs) leading to the formation of wortmannin-induced multivesicular compartments. Appearance of wortmannin-induced compartments was associated with depletion of TGN as revealed by electron microscopy. On the proteome level, wortmannin induced massive changes in protein abundance profiles. Wortmannin-sensitive proteins belonged to various functional classes. An inhibition of vacuolar trafficking by wortmannin was related to the downregulation of proteins targeted to the vacuole, as showed for vacuolar proteases. A small GTPase, RabA1d, which regulates vesicular trafficking at TGN, was identified as a new protein negatively affected by wortmannin. In addition, Sec14 was upregulated and PLD1 alpha was downregulated by wortmannin.
- MeSH
- androstadieny farmakologie MeSH
- Arabidopsis cytologie účinky léků metabolismus MeSH
- fosfolipasa D metabolismus MeSH
- kořeny rostlin cytologie účinky léků metabolismus MeSH
- multivezikulární tělíska účinky léků metabolismus MeSH
- proteiny huseníčku metabolismus MeSH
- proteiny přenášející fosfolipidy metabolismus MeSH
- proteom metabolismus MeSH
- rab proteiny vázající GTP metabolismus MeSH
- trans-Golgiho síť účinky léků metabolismus ultrastruktura MeSH
- transport proteinů účinky léků MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
