Resolving the homology-function relationship through comparative genomics of membrane-trafficking machinery and parasite cell biology

With advances in DNA sequencing technology, it is increasingly common and tractable to informatically look for genes of interest in the genomic databases of parasitic organisms and infer cellular states. Assignment of a putative gene function based on homology to functionally characterized genes in other organisms, though powerful, relies on the implicit assumption of functional homology, i.e. that orthology indicates conserved function. Eukaryotes reveal a dazzling array of cellular features and structural organization, suggesting a concomitant diversity in their underlying molecular machinery. Significantly, examples of novel functions for pre-existing or new paralogues are not uncommon. Do these examples undermine the basic assumption of functional homology, especially in parasitic protists, which are often highly derived? Here we examine the extent to which functional homology exists between organisms spanning the eukaryotic lineage. By comparing membrane trafficking proteins between parasitic protists and traditional model organisms, where direct functional evidence is available, we find that function is indeed largely conserved between orthologues, albeit with significant adaptation arising from the unique biological features within each lineage

RNAi-Based Suppressor Screens Reveal Genetic Interactions Between the CRL2LRR-1 E3-Ligase and the DNA Replication Machinery in Caenorhabditis elegans

Cullin-RING E3-Ligases (CRLs), the largest family of E3 ubiquitin-Ligases, regulate diverse cellular processes by promoting ubiquitination of target proteins. The evolutionarily conserved Leucine Rich Repeat protein 1 (LRR-1) is a substrate-recognition subunit of a CRL2LRR-1 E3-ligase. Here we provide genetic evidence supporting a role of this E3-enzyme in the maintenance of DNA replication integrity in Caenorhabditis elegans. Through RNAi-based suppressor screens of lrr-1(0) and cul-2(or209ts) mutants, we identified two genes encoding components of the GINS complex, which is part of the Cdc45-MCM-GINS (CMG) replicative helicase, as well as CDC-7 and MUS-101, which drives the assembly of the CMG helicase during DNA replication. In addition, we identified the core components of the ATR/ATL-1 DNA replication checkpoint pathway (MUS-101, ATL-1, CLSP-1, CHK-1). These results suggest that the CRL2LRR-1 E3-ligase acts to modify or degrade factor(s) that would otherwise misregulate the replisome, eventually leading to the activation of the DNA replication checkpoint

The ESCRT-III-Interacting Deubiquitinating Enzyme AMSH3 is Essential for Degradation of Ubiquitinated Membrane Proteins in Arabidopsis thaliana

Post-translational modification by ubiquitin plays a key role in the regulation of endocytic degradation in which ubiquitinated plasma membrane cargos are transported to the vacuole for degradation dependent on the ESCRT (endosomal sorting complex required for transport) machinery. Arabidopsis AMSH3 (ASSOCIATED MOLECULE WITH THE SH3 DOMAIN OF STAM 3) is a deubiquitinating enzyme that interacts with at least two subunits of the ESCRT-III machinery, VPS2.1 and VPS24.1. amsh3 null mutation causes seedling lethality, and amsh3 null mutants show defects in multiple intracellular trafficking pathways. In this study, we further analyzed the amsh3 mutant phenotype and showed that amsh3 accumulates membrane-associated ubiquitinated proteins, supporting the indication that AMSH3 functions in ubiquitin-mediated endocytic degradation. In accordance with this, an enzymatic inactive variant of AMSH3 inhibits the AvrPtoB-dependent endocytic degradation of CERK1 (CHITIN ELICITOR RECEPTOR KINASE 1). Furthermore, we showed that the interaction of AMSH3 with ESCRT-III is important for its function in planta. Together, our data indicate the importance of AMSH3 and the AMSH3-ESCRT-III interaction for deubiquitination and degradation of ubiquitinated membrane substrates in plants.publishe

MLN4924 Is an Efficient Inhibitor of NEDD8 Conjugation in Plants1[C][W][OA]

The conjugation of the ubiquitin-like modifier NEURAL PRECURSOR CELL-EXPRESSED DEVELOPMENTALLY DOWN-REGULATED PROTEIN8/RELATED TO UBIQUITIN1 (NEDD8/RUB1; neddylation) is best known as an important posttranslational modification of the cullin subunits of cullin-RING-type E3 ubiquitin ligases (CRLs). MLN4924 has recently been described as an inhibitor of NEDD8-ACTIVATING ENZYME1 (NAE1) in human. Here, we show that MLN4924 is also an effective and specific inhibitor of NAE1 enzymes from Arabidopsis (Arabidopsis thaliana) and other plant species. We found that MLN4924-treated wild-type seedlings have phenotypes that are highly similar to phenotypes of mutants with a partial defect in neddylation and that such neddylation-defective mutants are hypersensitive to MLN4924 treatment. We further found that MLN4924 efficiently blocks the neddylation of cullins in Arabidopsis and that MLN4924 thereby interferes with the degradation of CRL substrates and their downstream responses. MLN4924 treatments also induce characteristic phenotypes in tomato (Solanum lycopersicum), Cardamine hirsuta, and Brachypodium distachyon. Interestingly, MLN4924 also blocks the neddylation of a number of other NEDD8-modified proteins. In summary, we show that MLN4924 is a versatile and specific neddylation inhibitor that will be a useful tool to examine the role of NEDD8- and CRL-dependent processes in a wide range of plant species

The Deubiquitinating Enzyme AMSH3 Is Required for Intracellular Trafficking and Vacuole Biogenesis in Arabidopsis thaliana[C][W]

This study identifies AMSH3 as a major deubiquitinating enzyme in Arabidopsis that hydrolyzes ubiquitin chains in vitro and in vivo. It finds that AMSH3 is essential for proper vacuole biogenesis, trafficking from the Golgi to the vacuole, and the vacuolar trafficking of endocytosed cargo

The Deubiquitinating Enzyme AMSH1 and the ESCRT-III Subunit VPS2.1 are Required for Autophagic Degradation in Arabidopsis

In eukaryotes, posttranslational modification by ubiquitin regulates the activity and stability of many proteins and thus influences a variety of developmental processes as well as environmental responses. Ubiquitination also plays a critical role in intracellular trafficking by serving as a signal for endocytosis. We have previously shown that the Arabidopsis thaliana associated molecule with the SH3 domain of STAM3 (AMSH3) is a deubiquitinating enzyme (DUB) that interacts with endosomal complex required for transport-III (ESCRT-III) and is essential for intracellular transport and vacuole biogenesis. However, physiological functions of AMSH3 in the context of its ESCRT-III interaction are not well understood due to the severe seedling lethal phenotype of its null mutant. In this article, we show that Arabidopsis AMSH1, an AMSH3-related DUB, interacts with the ESCRT-III subunit vacuolar protein sorting2.1 (VPS2.1) and that impairment of both AMSH1 and VPS2.1 causes early senescence and hypersensitivity to artificial carbon starvation in the dark similar to previously reported autophagy mutants. Consistent with this, both mutants accumulate autophagosome markers and accumulate less autophagic bodies in the vacuole. Taken together, our results demonstrate that AMSH1 and the ESCRT-III-subunit VPS2.1 are important for autophagic degradation and autophagy-mediated physiological processes.publishe