HYPK promotes the activity of the Nα-acetyltransferase A complex to determine proteostasis of nonAc-X2/N-degron-containing proteins.

In humans, the Huntingtin yeast partner K (HYPK) binds to the ribosome-associated Nα-acetyltransferase A (NatA) complex that acetylates ~40% of the proteome in humans and Arabidopsis thaliana. However, the relevance of HsHYPK for determining the human N-acetylome is unclear. Here, we identify the AtHYPK protein as the first in vivo regulator of NatA activity in plants. AtHYPK physically interacts with the ribosome-anchoring subunit of NatA and promotes Nα-terminal acetylation of diverse NatA substrates. Loss-of-AtHYPK mutants are remarkably resistant to drought stress and strongly resemble the phenotype of NatA-depleted plants. The ectopic expression of HsHYPK rescues this phenotype. Combined transcriptomics, proteomics, and N-terminomics unravel that HYPK impairs plant metabolism and development, predominantly by regulating NatA activity. We demonstrate that HYPK is a critical regulator of global proteostasis by facilitating masking of the recently identified nonAc-X2/N-degron. This N-degron targets many nonacetylated NatA substrates for degradation by the ubiquitin-proteasome system.

Dual lysine and N-terminal acetyltransferases reveal the complexity underpinning protein acetylation.

Protein acetylation is a highly frequent protein modification. However, comparatively little is known about its enzymatic machinery. N-α-acetylation (NTA) and ε-lysine acetylation (KA) are known to be catalyzed by distinct families of enzymes (NATs and KATs, respectively), although the possibility that the same GCN5-related N-acetyltransferase (GNAT) can perform both functions has been debated. Here, we discovered a new family of plastid-localized GNATs, which possess a dual specificity. All characterized GNAT family members display a number of unique features. Quantitative mass spectrometry analyses revealed that these enzymes exhibit both distinct KA and relaxed NTA specificities. Furthermore, inactivation of GNAT2 leads to significant NTA or KA decreases of several plastid proteins, while proteins of other compartments were unaffected. The data indicate that these enzymes have specific protein targets and likely display partly redundant selectivity, increasing the robustness of the acetylation process in vivo. In summary, this study revealed a new layer of complexity in the machinery controlling this prevalent modification and suggests that other eukaryotic GNATs may also possess these previously underappreciated broader enzymatic activities.

The Arabidopsis Nα -acetyltransferase NAA60 locates to the plasma membrane and is vital for the high salt stress response.

In humans and plants, N-terminal acetylation plays a central role in protein homeostasis, affects 80% of proteins in the cytoplasm and is catalyzed by five ribosome-associated N-acetyltransferases (NatA-E). Humans also possess a Golgi-associated NatF (HsNAA60) that is essential for Golgi integrity. Remarkably, NAA60 is absent in fungi and has not been identified in plants. Here we identify and characterize the first plasma membrane-anchored post-translationally acting N-acetyltransferase AtNAA60 in the reference plant Arabidopsis thaliana by the combined application of reverse genetics, global proteomics, live-cell imaging, microscale thermophoresis, circular dichroism spectroscopy, nano-differential scanning fluorometry, intrinsic tryptophan fluorescence and X-ray crystallography. We demonstrate that AtNAA60, like HsNAA60, is membrane-localized in vivo by an α-helical membrane anchor at its C-terminus, but in contrast to HsNAA60, AtNAA60 localizes to the plasma membrane. The AtNAA60 crystal structure provides insights into substrate-binding, the broad substrate specificity and the catalytic mechanism probed by structure-based mutagenesis. Characterization of the NAA60 loss-of-function mutants (naa60-1 and naa60-2) uncovers a plasma membrane-localized substrate of AtNAA60 and the importance of NAA60 during high salt stress. Our findings provide evidence for the plant-specific evolution of a plasma membrane-anchored N-acetyltransferase that is vital for adaptation to stress.

NAA50 Is an Enzymatically Active N α-Acetyltransferase That Is Crucial for Development and Regulation of Stress Responses.

Nα-terminal acetylation (NTA) is a prevalent protein modification in eukaryotes. In plants, the biological function of NTA remains enigmatic. The dominant N-acetyltransferase (Nat) in Arabidopsis (Arabidopsis thaliana) is NatA, which cotranslationally catalyzes acetylation of ∼40% of the proteome. The core NatA complex consists of the catalytic subunit NAA10 and the ribosome-anchoring subunit NAA15. In human (Homo sapiens), fruit fly (Drosophila melanogaster), and yeast (Saccharomyces cerevisiae), this core NatA complex interacts with NAA50 to form the NatE complex. While in metazoa, NAA50 has N-acetyltransferase activity, yeast NAA50 is catalytically inactive and positions NatA at the ribosome tunnel exit. Here, we report the identification and characterization of Arabidopsis NAA50 (AT5G11340). Consistent with its putative function as a cotranslationally acting Nat, AtNAA50-EYFP localized to the cytosol and the endoplasmic reticulum but also to the nuclei. We demonstrate that purified AtNAA50 displays Nα-terminal acetyltransferase and lysine-ε-autoacetyltransferase activity in vitro. Global N-acetylome profiling of Escherichia coli cells expressing AtNAA50 revealed conservation of NatE substrate specificity between plants and humans. Unlike the embryo-lethal phenotype caused by the absence of AtNAA10 and AtNAA15, loss of NAA50 expression resulted in severe growth retardation and infertility in two Arabidopsis transfer DNA insertion lines (naa50-1 and naa50-2). The phenotype of naa50-2 was rescued by the expression of HsNAA50 or AtNAA50. In contrast, the inactive ScNAA50 failed to complement naa50-2 Remarkably, loss of NAA50 expression did not affect NTA of known NatA substrates and caused the accumulation of proteins involved in stress responses. Overall, our results emphasize a relevant role of AtNAA50 in plant defense and development, which is independent of the essential NatA activity.

NatB-Mediated N-Terminal Acetylation Affects Growth and Biotic Stress Responses.

N∝-terminal acetylation (NTA) is one of the most abundant protein modifications in eukaryotes. In humans, NTA is catalyzed by seven Nα-acetyltransferases (NatA-F and NatH). Remarkably, the plant Nat machinery and its biological relevance remain poorly understood, although NTA has gained recognition as a key regulator of crucial processes such as protein turnover, protein-protein interaction, and protein targeting. In this study, we combined in vitro assays, reverse genetics, quantitative N-terminomics, transcriptomics, and physiological assays to characterize the Arabidopsis (Arabidopsis thaliana) NatB complex. We show that the plant NatB catalytic (NAA20) and auxiliary subunit (NAA25) form a stable heterodimeric complex that accepts canonical NatB-type substrates in vitro. In planta, NatB complex formation was essential for enzymatic activity. Depletion of NatB subunits to 30% of the wild-type level in three Arabidopsis T-DNA insertion mutants (naa20-1, naa20-2, and naa25-1) caused a 50% decrease in plant growth. A complementation approach revealed functional conservation between plant and human catalytic NatB subunits, whereas yeast NAA20 failed to complement naa20-1 Quantitative N-terminomics of approximately 1000 peptides identified 32 bona fide substrates of the plant NatB complex. In vivo, NatB was seen to preferentially acetylate N termini starting with the initiator Met followed by acidic amino acids and contributed 20% of the acetylation marks in the detected plant proteome. Global transcriptome and proteome analyses of NatB-depleted mutants suggested a function of NatB in multiple stress responses. Indeed, loss of NatB function, but not NatA, increased plant sensitivity toward osmotic and high-salt stress, indicating that NatB is required for tolerance of these abiotic stressors.

Structural and genomic decoding of human and plant myristoylomes reveals a definitive recognition pattern.

An organism's entire protein modification repertoire has yet to be comprehensively mapped. N-myristoylation (MYR) is a crucial eukaryotic N-terminal protein modification. Here we mapped complete Homo sapiens and Arabidopsis thaliana myristoylomes. The crystal structures of human modifier NMT1 complexed with reactive and nonreactive target-mimicking peptide ligands revealed unexpected binding clefts and a modifier recognition pattern. This information allowed integrated mapping of myristoylomes using peptide macroarrays, dedicated prediction algorithms, and in vivo mass spectrometry. Global MYR profiling at the genomic scale identified over a thousand novel, heterogeneous targets in both organisms. Surprisingly, MYR involved a non-negligible set of overlapping targets with N-acetylation, and the sequence signature marks for a third proximal acylation-S-palmitoylation-were genomically imprinted, allowing recognition of sequences exhibiting both acylations. Together, the data extend the N-end rule concept for Gly-starting proteins to subcellular compartmentalization and reveal the main neighbors influencing protein modification profiles and consequent cell fate.

The C-terminal residue of phage Vp16 PDF, the smallest peptide deformylase, acts as an offset element locking the active conformation.

Prokaryotic proteins must be deformylated before the removal of their first methionine. Peptide deformylase (PDF) is indispensable and guarantees this mechanism. Recent metagenomics studies revealed new idiosyncratic PDF forms as the most abundant family of viral sequences. Little is known regarding these viral PDFs, including the capacity of the corresponding encoded proteins to ensure deformylase activity. We provide here the first evidence that viral PDFs, including the shortest PDF identified to date, Vp16 PDF, display deformylase activity in vivo, despite the absence of the key ribosome-interacting C-terminal region. Moreover, characterization of phage Vp16 PDF underscores unexpected structural and molecular features with the C-terminal Isoleucine residue significantly contributing to deformylase activity both in vitro and in vivo. This residue fully compensates for the absence of the usual long C-domain. Taken together, these data elucidate an unexpected mechanism of enzyme natural evolution and adaptation within viral sequences.

EnCOUNTer: a parsing tool to uncover the mature N-terminus of organelle-targeted proteins in complex samples.

BACKGROUND: Characterization of mature protein N-termini by large scale proteomics is challenging. This is especially true for proteins undergoing cleavage of transit peptides when they are targeted to specific organelles, such as mitochondria or chloroplast. Protein neo-N-termini can be located up to 100-150 amino acids downstream from the initiator methionine and are not easily predictable. Although some bioinformatics tools are available, they usually require extensive manual validation to identify the exact N-terminal position. The situation becomes even more complex when post-translational modifications take place at the neo-N-terminus. Although N-terminal acetylation occurs mostly in the cytosol, it is also observed in some organelles such as chloroplast. To date, no bioinformatics tool is available to define mature protein starting positions, the associated N-terminus acetylation status and/or yield for each proteoform. In this context, we have developed the EnCOUNTer tool (i) to score all characterized peptides using discriminating parameters to identify bona fide mature protein N-termini and (ii) to determine the N-terminus acetylation yield of the most reliable ones. RESULTS: Based on large scale proteomics analyses using the SILProNAQ methodology, tandem mass spectrometry favoured the characterization of thousands of peptides. Data processing using the EnCOUNTer tool provided an efficient and rapid way to extract the most reliable mature protein N-termini. Selected peptides were subjected to N-terminus acetylation yield determination. In an A. thaliana cell lysate, 1232 distinct proteotypic N-termini were characterized of which 648 were located at the predicted protein N-terminus (position 1/2) and 584 were located further downstream (starting at position > 2). A large number of these N-termini were associated with various well-defined maturation processes occurring on organelle-targeted proteins (mitochondria, chloroplast and peroxisome), secreted proteins or membrane-targeted proteins. It was also possible to highlight some protein alternative starts, splicing variants or erroneous protein sequence predictions. CONCLUSIONS: The EnCOUNTer tool provides a unique way to extract accurately the most relevant mature proteins N-terminal peptides from large scale experimental datasets. Such data processing allows the identification of the exact N-terminus position and the associated acetylation yield.

SILProNAQ: A Convenient Approach for Proteome-Wide Analysis of Protein N-Termini and N-Terminal Acetylation Quantitation.

Protein N-terminal modifications have recently been involved in overall proteostasis through their impact on cell fate and protein life time. This explains the development of new approaches to characterize more precisely the N-terminal end of mature proteins. Although few approaches are available to perform N-terminal enrichment based on positive or negative discriminations, these methods are usually restricted to the enrichment in N-terminal peptides and their characterization by mass spectrometry. Recent investigation highlights both (1) the knowledge of the N-terminal acetylation status of most cytosolic proteins and (2) post-translational addition of this modification on the N-terminus of nuclear coded chloroplast proteins imported in the plastid and after the cleavage of the transit peptide. The workflow involves stable isotope labeling to assess N-acetylation rates followed by Strong Cation eXchange (SCX ) fractionation of the samples to provide protein N-terminal enriched fractions. Combined with mass spectrometry analyses, the technology finally requires extensive data processing. This last step aims first at discriminating the most relevant mature N-termini from the characterized peptides, next at determining its experimental position and then at calculating the N-terminal acetylation yield. Stable-Isotope Protein N-terminal Acetylation Quantification (SILProNAQ) is a complete workflow combining wet-lab techniques together with dry-lab processing to determine the N-terminal acetylation yield of mature proteins for a clearly defined localization.

MetAP1 and MetAP2 drive cell selectivity for a potent anti-cancer agent in synergy, by controlling glutathione redox state.

Fumagillin and its derivatives are therapeutically useful because they can decrease cancer progression. The specific molecular target of fumagillin is methionine aminopeptidase 2 (MetAP2), one of the two MetAPs present in the cytosol. MetAPs catalyze N-terminal methionine excision (NME), an essential pathway of cotranslational protein maturation. To date, it remains unclear the respective contribution of MetAP1 and MetAP2 to the NME process in vivo and why MetAP2 inhibition causes cell cycle arrest only in a subset of cells. Here, we performed a global characterization of the N-terminal methionine excision pathway and the inhibition of MetAP2 by fumagillin in a number of lines, including cancer cell lines. Large-scale N-terminus profiling in cells responsive and unresponsive to fumagillin treatment revealed that both MetAPs were required in vivo for M[VT]X-targets and, possibly, for lower-level M[G]X-targets. Interestingly, we found that the responsiveness of the cell lines to fumagillin was correlated with the ability of the cells to modulate their glutathione homeostasis. Indeed, alterations to glutathione status were observed in fumagillin-sensitive cells but not in cells unresponsive to this agent. Proteo-transcriptomic analyses revealed that both MetAP1 and MetAP2 accumulated in a cell-specific manner and that cell sensitivity to fumagillin was related to the levels of these MetAPs, particularly MetAP1. We suggest that MetAP1 levels could be routinely checked in several types of tumor and used as a prognostic marker for predicting the response to treatments inhibiting MetAP2.

Downregulation of N-terminal acetylation triggers ABA-mediated drought responses in Arabidopsis.

N-terminal acetylation (NTA) catalysed by N-terminal acetyltransferases (Nats) is among the most common protein modifications in eukaryotes, but its significance is still enigmatic. Here we characterize the plant NatA complex and reveal evolutionary conservation of NatA biochemical properties in higher eukaryotes and uncover specific and essential functions of NatA for development, biosynthetic pathways and stress responses in plants. We show that NTA decreases significantly after drought stress, and NatA abundance is rapidly downregulated by the phytohormone abscisic acid. Accordingly, transgenic downregulation of NatA induces the drought stress response and results in strikingly drought resistant plants. Thus, we propose that NTA by the NatA complex acts as a cellular surveillance mechanism during stress and that imprinting of the proteome by NatA is an important switch for the control of metabolism, development and cellular stress responses downstream of abscisic acid.

Molecular identification and functional characterization of the first Nα-acetyltransferase in plastids by global acetylome profiling.

Protein N(α) -terminal acetylation represents one of the most abundant protein modifications of higher eukaryotes. In humans, six N(α) -acetyltransferases (Nats) are responsible for the acetylation of approximately 80% of the cytosolic proteins. N-terminal protein acetylation has not been evidenced in organelles of metazoans, but in higher plants is a widespread modification not only in the cytosol but also in the chloroplast. In this study, we identify and characterize the first organellar-localized Nat in eukaryotes. A primary sequence-based search in Arabidopsis thaliana revealed seven putatively plastid-localized Nats of which AT2G39000 (AtNAA70) showed the highest conservation of the acetyl-CoA binding pocket. The chloroplastic localization of AtNAA70 was demonstrated by transient expression of AtNAA70:YFP in Arabidopsis mesophyll protoplasts. Homology modeling uncovered a significant conservation of tertiary structural elements between human HsNAA50 and AtNAA70. The in vivo acetylation activity of AtNAA70 was demonstrated on a number of distinct protein N(α) -termini with a newly established global acetylome profiling test after expression of AtNAA70 in E. coli. AtNAA70 predominately acetylated proteins starting with M, A, S and T, providing an explanation for most protein N-termini acetylation events found in chloroplasts. Like HsNAA50, AtNAA70 displays N(ε) -acetyltransferase activity on three internal lysine residues. All MS data have been deposited in the ProteomeXchange with identifier PXD001947 (http://proteomecentral.proteomexchange.org/dataset/PXD001947).

Two N-terminal acetyltransferases antagonistically regulate the stability of a nod-like receptor in Arabidopsis.

Nod-like receptors (NLRs) serve as immune receptors in plants and animals. The stability of NLRs is tightly regulated, though its mechanism is not well understood. Here, we show the crucial impact of N-terminal acetylation on the turnover of one plant NLR, Suppressor of NPR1, Constitutive 1 (SNC1), in Arabidopsis thaliana. Genetic and biochemical analyses of SNC1 uncovered its multilayered regulation by different N-terminal acetyltransferase (Nat) complexes. SNC1 exhibits a few distinct N-terminal isoforms generated through alternative initiation and N-terminal acetylation. Its first Met is acetylated by N-terminal acetyltransferase complex A (NatA), while the second Met is acetylated by N-terminal acetyltransferase complex B (NatB). Unexpectedly, the NatA-mediated acetylation serves as a degradation signal, while NatB-mediated acetylation stabilizes the NLR protein, thus revealing antagonistic N-terminal acetylation of a single protein substrate. Moreover, NatA also contributes to the turnover of another NLR, RESISTANCE TO P. syringae pv maculicola 1. The intricate regulation of protein stability by Nats is speculated to provide flexibility for the target protein in maintaining its homeostasis.

Proteome-wide analysis of the amino terminal status of Escherichia coli proteins at the steady-state and upon deformylation inhibition.

A proteome wide analysis was performed in Escherichia coli to identify the impact on protein N-termini of actinonin, an antibiotic specifically inhibiting peptide deformylase (PDF). A strategy and tool suite (SILProNaQ) was employed to provide large-scale quantitation of N-terminal modifications. In control conditions, more than 1000 unique N-termini were identified with 56% showing initiator methionine removal. Additional modifications corresponded to partial or complete Nα-acetylation (10%) and N-formyl retention (5%). Among the proteins undergoing these N-terminal modifications, 140 unique N-termini from translocated membrane proteins were highlighted. The very early time-course impact of actinonin was followed after addition of bacteriostatic concentrations of the drug. Under these conditions, 26% of all proteins did not undergo deformylation any longer after 10 min, a value reaching more than 60% of all characterized proteins after 40 min of treatment. The N-formylation ratio measured on individual proteins increased with the same trend. Upon early PDF inhibition, two major categories of proteins retained their N-formyl group: a large number of inner membrane proteins and many proteins involved in protein synthesis including factors assisting the nascent chains in early cotranslational events. All MS data have been deposited in the ProteomeXchange with identifiers PXD001979, PXD002012 and PXD001983 (http://proteomecentral.proteomexchange.org/dataset/PXD001979, http://proteomecentral.proteomexchange.org/dataset/PXD002012 and http://proteomecentral.proteomexchange.org/dataset/PXD001983).

RPM-1 uses both ubiquitin ligase and phosphatase-based mechanisms to regulate DLK-1 during neuronal development.

The Pam/Highwire/RPM-1 (PHR) proteins are key regulators of neuronal development that function in axon extension and guidance, termination of axon outgrowth, and synapse formation. Outside of development, the PHR proteins also regulate axon regeneration and Wallerian degeneration. The PHR proteins function in part by acting as ubiquitin ligases that degrade the Dual Leucine zipper-bearing Kinase (DLK). Here, we show that the Caenorhabditis elegans PHR protein, Regulator of Presynaptic Morphology 1 (RPM-1), also utilizes a phosphatase-based mechanism to regulate DLK-1. Using mass spectrometry, we identified Protein Phosphatase Magnesium/Manganese dependent 2 (PPM-2) as a novel RPM-1 binding protein. Genetic, transgenic, and biochemical studies indicated that PPM-2 functions coordinately with the ubiquitin ligase activity of RPM-1 and the F-box protein FSN-1 to negatively regulate DLK-1. PPM-2 acts on S874 of DLK-1, a residue implicated in regulation of DLK-1 binding to a short, inhibitory isoform of DLK-1 (DLK-1S). Our study demonstrates that PHR proteins function through both phosphatase and ubiquitin ligase mechanisms to inhibit DLK. Thus, PHR proteins are potentially more accurate and sensitive regulators of DLK than originally thought. Our results also highlight an important and expanding role for the PP2C phosphatase family in neuronal development.

Extracellular signal-regulated kinase regulates RhoA activation and tumor cell plasticity by inhibiting guanine exchange factor H1 activity.

In certain Ras mutant cell lines, the inhibition of extracellular signal-regulated kinase (ERK) signaling increases RhoA activity and inhibits cell motility, which was attributed to a decrease in Fra-1 levels. Here we report a Fra-1-independent augmentation of RhoA signaling during short-term inhibition of ERK signaling. Using mass spectrometry-based proteomics, we identified guanine exchange factor H1 (GEF-H1) as mediating this effect. ERK binds to the Rho exchange factor GEF-H1 and phosphorylates it on S959, causing inhibition of GEF-H1 activity and a consequent decrease in RhoA activity. Knockdown experiments and expression of a nonphosphorylatable S959A GEF-H1 mutant showed that this site is crucial in regulating cell motility and invasiveness. Thus, we identified GEF-H1 as a critical ERK effector that regulates motility, cell morphology, and invasiveness.

Influence of various endogenous and artefact modifications on large-scale proteomics analysis.

RATIONALE: Some large-scale proteomics studies in which strong cation exchange chromatography has been applied are used to determine proteomes and post-translational modification dynamics. Although such datasets favour the characterisation of thousands of modified peptides, e.g., phosphorylated and N-α-acetylated, a large fraction of the acquired spectra remain unexplained by standard proteomics approaches. Thus, advanced data processing allows characterisation of a significant part of these unassigned spectra. METHODS: Our recent investigation of the N-α-acetylation status of plant proteins gave a dataset of choice to investigate further the in-depth characterisation of peptide modifications using Mascot tools associated with relevant validation processes. Such an approach allows to target frequently occurring modifications such as methionine oxidation, phosphorylation or N-α-acetylation, but also the less usual peptide cationisation. Finally, this dataset offers the unique opportunity to determine the overall influence of some of these modifications on the identification score. RESULTS: Although methionine oxidation has no influence and tends to favour the characterisation of protein N-terminal peptides, peptide alkalinisation shows an adverse effect on peptide average score. Nevertheless, peptide cationisation appears to favour the characterisation of protein C-terminal peptides with a limited to no direct influence on the identification score. Unexpectedly, our investigation reveals the unfortunate combination of the molecular weight of N-α-acetylation and potassium cation that mimics the mass increment of a phosphorylation group. CONCLUSIONS: Since these characterisations rely upon computational treatment associated with statistical validation approaches such as 'False discovery rates' calculation or post-translational modification position validation, our investigation highlights the limitation of such treatment which is biased by the initial searched hypotheses.

Identification of protein interfaces between α-synuclein, the principal component of Lewy bodies in Parkinson disease, and the molecular chaperones human Hsc70 and the yeast Ssa1p.

Fibrillar α-synuclein (α-Syn) is the principal component of Lewy bodies, which are evident in individuals affected by Parkinson disease (PD). This neuropathologic form of α-Syn plays a central role in PD progression as it has been shown to propagate between neurons. Tools that interfere with α-Syn assembly or change the physicochemical properties of the fibrils have potential therapeutic properties as they may be sufficient to interfere with and/or halt cell-to-cell transmission and the systematic spread of α-Syn assemblies within the central nervous system. Vertebrate molecular chaperones from the constitutive/heat-inducible heat shock protein 70 (Hsc/p70) family have been shown to hinder the assembly of soluble α-Syn into fibrils and to bind to the fibrils and very significantly reduce their toxicity. To understand how Hsc70 family members sequester soluble α-Syn, we set up experiments to identify the molecular chaperone-α-Syn surface interfaces. We cross-linked human Hsc70 and its yeast homologue Ssa1p and α-Syn using a chemical cross-linker and mapped the Hsc70- and Ssa1p-α-Syn interface. We show that the client binding domain of Hsc70 and Ssa1p binds two regions within α-Syn similar to a tweezer, with the first spanning residues 10-45 and the second spanning residues 97-102. Our findings define what is necessary and sufficient for engineering Hsc70- and Ssa1p-derived polypeptide with minichaperone properties with a potential as therapeutic agents in Parkinson disease through their ability to affect α-Syn assembly and/or toxicity.

RAE-1, a novel PHR binding protein, is required for axon termination and synapse formation in Caenorhabditis elegans.

Previous studies in Caenorhabditis elegans showed that RPM-1 (Regulator of Presynaptic Morphology-1) regulates axon termination and synapse formation. To understand the mechanism of how rpm-1 functions, we have used mass spectrometry to identify RPM-1 binding proteins, and have identified RAE-1 (RNA Export protein-1) as an evolutionarily conserved binding partner. We define a RAE-1 binding region in RPM-1, and show that this binding interaction is conserved and also occurs between Rae1 and the human ortholog of RPM-1 called Pam (protein associated with Myc). rae-1 loss of function causes similar axon and synapse defects, and synergizes genetically with two other RPM-1 binding proteins, GLO-4 and FSN-1. Further, we show that RAE-1 colocalizes with RPM-1 in neurons, and that rae-1 functions downstream of rpm-1. These studies establish a novel postmitotic function for rae-1 in neuronal development.