Epitope tag immunoblotting represents a routine method for targeted surveys of protein abundance and expression. Especially for microproteins, defined by an arbitrary cutoff of 100 amino acids in length, blotting-based approaches are indispensable as the small size of microproteins often renders them elusive to mass spectrometry. Nonetheless, the blotting of microproteins introduces a set of technical challenges, leading to microprotein losses, which significantly affect the sensitivity of blotting-based approaches like epitope tag immunoblotting.We introduce HiBiT blotting, an antibody-free luminescent detection method for HiBiT-tagged proteins, offering an alternative blotting-based protein detection method to improve microprotein analysis. The availability of an anti-HiBiT antibody enabled a comparative analysis of HiBiT versus classical epitope tag immunoblotting, overall demonstrating the superior sensitivity of HiBiT blotting in detecting HiBiT-tagged microproteins. By offering a more direct and sensitive approach for small protein analysis, HiBiT blotting represents a substantial contribution to the field, enabling the effective study of microproteins and addressing the longstanding challenge of their detection.

ABSTRACT Type III secretion systems (T3SSs) enable Salmonella to modulate host cell biology by delivering a diverse repertoire of effector proteins. Yet, the temporal dynamics of effector translocation during prolonged infection remain poorly defined. Here, we establish a systematic real-time framework for quantifying effector secretion and translocation by combining endogenous HiBiT-tagging with split NanoLuc-based detection. Using this approach, we monitored the translocation kinetics of all 39 currently annotated type III effectors (T3Es) in Salmonella enterica serovar Typhimurium SL1344 over a 24-hour infection of epithelial cells. This comprehensive analysis revealed temporally distinct translocation patterns, widespread mid- and late-stage effector delivery, and a quantitatively resolved functional overlap between T3SS-1 and T3SS-2-mediated secretion than previously appreciated. These findings provide a more refined view of effector deployment throughout infection and illuminate previously overlooked aspects of effector dynamics. Altogether, this work offers a detailed temporal map of effector dynamics during epithelial infection and establishes a scalable strategy for dissecting secretion system function across host-pathogen systems.

Biotin identification (BioID) is an interactomics approach that utilizes proximity labelling to map the local interactome or proxeome of proteins within a cell. This study applies BioID to investigate proteins proximal to NAA60 (N-alpha-acetyltransferase 60), an N-terminal acetyltransferase (NAT) of pathological significance in human disease, characterized by its unique Golgi localization. NAA60 is known to N-terminally acetylate transmembrane proteins that present their N-terminus on the cytosolic face of the membrane, and its involvement in maintaining Golgi structure has previously been established. Using a stable cell-line expressing an NAA60-BirA* fusion protein, we isolated biotinylated proteins through streptavidin affinity purification. Mass spectrometry analysis revealed over 100 proximal partners of NAA60, enriched in proteins localized on the trans-side of the Golgi apparatus. High-confidence proximity interactors included golgins and GRASP proteins, essential for Golgi integrity. Considering the transmembrane nature of NAA60, the identification of biotinylated peptides inferred the topology of transmembrane protein interactors within the secretory pathway. Subsequent suborganellar localization analysis revealed a more prominent medial/trans-Golgi localization of NAA60. Our findings underscore the role of NAA60 and its interactors in maintaining Golgi structural integrity and highlight the effectiveness of BioID in generating critical protein topology data, invaluable for enhancing the prediction of protein topology within cellular compartments.

Root-knot nematodes ( Meloidogyne spp.) are obligatory plant root parasites whose effector proteins play a critical role in suppressing plant immunity. However, the effectors direct host targets and underlying molecular mechanisms remain poorly understood. Using TurboID-mediated proximity labeling in tomato ( Solanum lycopersicum ) hairy roots, we identified an interaction between the nematode effector Mj-MSP18 and the tomato BRASSINOSTEROID-SIGNALING KINASE 7 (Sl-BSK7). Yeast two-hybrid (Y2H) assays confirmed that this interaction is conserved in Arabidopsis thaliana and Nicotiana benthamiana . Additionally, yeast three-hybrid and luciferase complementation assays demonstrated that Mj-MSP18 disrupts the interaction between BSK7/8 and FLS2 in both yeast and in planta . Given that the BSK7/8–FLS2 interaction is essential for flg22-induced pattern-triggered immunity (PTI), this disruption likely accounts for the suppression of reactive oxygen species (ROS) production and callose deposition observed upon transient expression of Mj-MSP18 in flg22-treated N. benthamiana leaves. Correspondingly, Arabidopsis and tomato bsk7 mutants exhibited increased susceptibility to root-knot nematode infection. Furthermore, RNA-seq analysis of tomato hairy roots expressing Mj-MSP18 revealed extensive transcriptional reprogramming, including the downregulation of defence-related genes and hydrogen peroxide response pathways. In addition, Y2H screening identified Sl-MYB and Sl-MYC2 as additional interactors, linking Mj-MSP18 to phytohormone biosynthesis, particularly the brassinosteroid (BR) and salicylic acid (SA) pathways, as validated by targeted metabolite analysis. The conservation of Mj-MSP18 across Meloidogyne species suggests a broadly conserved mechanism for host immune suppression and phytohormone modulation. ### Competing Interest Statement The authors have declared no competing interest. Special Research Fund of Ghent University Research Foundation - Flanders, https://ror.org/03qtxy027, G045921N Czech Science Foundation, 22-17435S China Scholarship Council, https://ror.org/04atp4p48

Since the initial use of BirA* for proximity-dependent biotin identification (BioID), researchers have explored variations of, and alternatives to, this specific biotin ligase enzyme. The application of these biotin ligase modules across diverse model systems has shown advancements in biotinylation efficiency and reduced labeling time. However, these improvements have also introduced challenges, such as increased background labeling. Bacterial BioID, a relatively recent development, is currently limited to studying bait proteins using miniTurbo- and TurboID-based fusions. So far, a comprehensive comparative analysis of the various promiscuous biotin ligases (PBLs) has been reported for only one bacterial species. In this chapter, we present a practical guide for selecting the most effective PBL tailored to the specific requirements of the bait and its corresponding model system for BioID-based interactome mapping in bacteria. Using the Salmonella enterica serovar Typhimurium type III effector protein SopB as a case study, we highlight a pioneering exploration of endogenous effector interactions directly within bacterial cells.

N-terminal proteoforms (Nt-proteoforms), which arise through alternative translation initiation, exhibit variation at their N-termini that can influence subcellular localization, protein stability, and functional roles within multiprotein complexes. Advances in riboproteogenomics, particularly in ribosome profiling, have highlighted the significance of the alternative proteome in bacteria. However, Nt-proteoforms remain largely underexplored, emphasizing the need for detailed protein-protein interaction (PPI) studies to elucidate their potentially differential biological roles.Mass spectrometry-based proximity labeling techniques offer a high-throughput method for PPI screening. Here, we present a multiplexed recombineering approach that enables the genomic integration of a promiscuous biotin ligase (PBL) via double-stranded DNA (dsDNA) recombineering while simultaneously allowing the targeted mutation of specific translation initiation sites (TIS) using single-stranded DNA (ssDNA) oligos through oligo-mediated allelic replacement (OMAR). This multiplexed recombineering approach facilitates endogenous BioID tagging and the identification of Nt-proteoform-specific proxeomes.

Proteomics has become a powerful approach for the identification and characterization of type III effectors (T3Es). Members of the Ralstonia solanacearum species complex (RSSC) deploy T3Es to manipulate host cells and to promote root infection of, among others, a wide range of solanaceous plants such as tomato, potato, and tobacco. Here, we used TurboID-mediated proximity labeling (PL) in tomato hairy root cultures to explore the proxeomes of the core RSSC T3Es RipU, RipD, and RipB. The RipU proxeome was enriched for multiple protein kinases, suggesting a potential impact on the two branches of the plant immune surveillance system, being the membrane-localized PAMP-triggered immunity (PTI) and the RIN4-dependent effector-triggered immunity (ETI) complexes. In agreement, a transcriptomics analysis in tomato revealed the potential involvement of RipU in modulating reactive oxygen species (ROS) signaling. The proxeome of RipB was putatively enriched for mitochondrial and chloroplast proteins and that of RipD for proteins potentially involved in the endomembrane system. Together, our results demonstrate that TurboID-PL in tomato hairy roots represents a promising tool to study Ralstonia T3E targets and functioning and that it can unravel potential host processes that can be hijacked by the bacterial pathogen.

Bacterial genome annotations are continuously refined with the advent of novel techniques. Ribosome profiling, or Ribo-seq, utilizing next-generation sequencing to link genomic regions with translational activity, has uncovered numerous small open reading frames (sORFs) - arbitrarily defined as ORFs no longer than 300 base pairs - as a generally under-annotated class of genomic elements in both eukaryotic and prokaryotic genomes. While sORFs can function as regulatory elements, they may also translate into small proteins (equal to or shorter than 100 amino acids), classified as sORF-encoded polypeptides (SEPs). The inherent limitations of ribosome profiling necessitate the experimental validation of predicted (s)ORFs at the protein expression level. However, the small size and unique biochemical characteristics of SEPs pose significant challenges for traditional protein detection methods, like mass spectrometry and immunoblotting. In this study, we introduce HiBiT blotting, a luminescent, complementation-based protein detection method, as a highly sensitive alternative to antibody-based immunoblotting for investigating SEP expression in Salmonella enterica (serovar) Typhimurium (S. Typhimurium) at endogenous levels. We demonstrate its complementarity to mass spectrometry as an expression validation tool. Additionally, employing a biochemical fractionation approach, we determined the subcellular localization of validated S. Typhimurium SEPs, initiating exploration into the functional aspects of these SEPs, and proposed enrichment strategies that may facilitate future experimental validation of Ribo-seq-predicted sORFs.

The pathogenic bacterium Salmonella survives and replicates in host cells within a Salmonella -containing vacuole (SCV). To build this niche, Salmonella uses secreted effectors, such as SopB, a phosphoinositide phosphotransferase that modifies host phospholipid fluxes at the plasma membrane and SCV. These lipid modifications impact host protein recruitment and activity, implicating SopB in bacterial internalization, SCV biogenesis, and inflammatory signaling. Yet, interactions of SopB with host proteins have remained ill-defined. Here, we employ the unique Virotrap mass spectrometry-based technology to identify a new set of SopB-associated host proteins. We demonstrate the direct interaction of SopB with the ubiquitin-binding domains of the ESCRT-0 subunit HGS, and we show that SopB promotes ESCRT-0 recruitment at the SCV where they colocalize. As the ESCRT machinery plays a central role in cargo sorting and membrane remodeling, we propose a new SopB-dependent mechanism by which Salmonella controls host membrane dynamics. ### Competing Interest Statement The authors have declared no competing interest.

In bacteria, cell poles function as subcellular compartments where proteins localize during specific lifecycle stages, orchestrated by polar "hub" proteins. Whereas most described bacteria inherit an "old" pole from the mother cell and a "new" pole from cell division, generating cell asymmetry at birth, non-binary division poses challenges for establishing cell polarity, particularly for daughter cells inheriting only new poles. We investigated polarity dynamics in the obligate predatory bacterium Bdellovibrio bacteriovorus, proliferating through filamentous growth followed by non-binary division within prey bacteria. Monitoring the subcellular localization of two proteins known as polar hubs in other species, RomR and DivIVA, revealed RomR as an early polarity marker in B. bacteriovorus. RomR already marks the future anterior poles of the progeny during the predator's growth phase, during a precise period closely following the onset of divisome assembly and the end of chromosome segregation. In contrast to RomR's stable unipolar localization in the progeny, DivIVA exhibits a dynamic pole-to-pole localization. This behavior changes shortly before the division of the elongated predator cell, where DivIVA accumulates at all septa and both poles. In vivo protein interaction networks for DivIVA and RomR, mapped through endogenous miniTurbo-based proximity labeling, further underscore their distinct roles in cell polarization and reinforce the importance of the anterior "invasive" cell pole in prey-predator interactions. Our work also emphasizes the precise spatiotemporal order of cellular processes underlying B. bacteriovorus proliferation, offering insights into the subcellular organization of bacteria with filamentous growth and non-binary division. IMPORTANCE In bacteria, cell poles are crucial areas where "hub" proteins orchestrate lifecycle events through interactions with multiple partners at specific times. While most bacteria exhibit one "old" and one "new" pole, inherited from the previous division event, setting polar identity poses challenges in bacteria with non-binary division. This study explores polar proteins in the predatory bacterium Bdellovibrio bacteriovorus, which undergoes filamentous growth followed by non-binary division inside another bacterium. Our research reveals distinct localization dynamics of the polar proteins RomR and DivIVA, highlighting RomR as an early "hub" marking polar identity in the filamentous mother cell. Using miniTurbo-based proximity labeling, we uncovered their unique protein networks. Overall, our work provides new insights into the cell polarity in non-binary dividing bacteria.

Citrobacter rodentium is an enteropathogen that causes intestinal inflammatory responses in mice reminiscent of the pathology provoked by enteropathogenic and enterohemorrhagic Escherichia coli infections in humans. C. rodentium expresses various virulence factors that target specific signaling proteins involved in executing apoptotic, necroptotic and pyroptotic cell death, suggesting that each of these distinct cell death modes performs essential host defense functions that the pathogen aims to disturb. However, the relative contributions of apoptosis, necroptosis and pyroptosis in protecting the host against C. rodentium have not been elucidated. Here we used mice with single or combined deficiencies in essential signaling proteins controlling apoptotic, necroptotic or pyroptotic cell death to reveal the roles of these cell death modes in host defense against C. rodentium. Gastrointestinal C. rodentium infections in mice lacking GSDMD and/or MLKL showed that both pyroptosis and necroptosis were dispensable for pathogen clearance. In contrast, while RIPK3-deficient mice showed normal C. rodentium clearance, mice with combined caspase-8 and RIPK3 deficiencies failed to clear intestinal pathogen loads. Although this demonstrated a crucial role for caspase-8 signaling in establishing intestinal host defense, Casp8-/-Ripk3-/- mice remained capable of preventing systemic pathogen persistence. This systemic host defense relied on inflammasome signaling, as Casp8-/-Ripk3-/- mice with combined caspase-1 and -11 deletion succumbed to C. rodentium infection. Interestingly, although it is known that C. rodentium can activate the non-canonical caspase-11 inflammasome, selectively disabling canonical inflammasome signaling by single caspase-1 deletion sufficed to render Casp8-/-Ripk3-/- mice vulnerable to C. rodentium-induced lethality. Moreover, Casp8-/-Ripk3-/- mice lacking GSDMD survived a C. rodentium infection, suggesting that pyroptosis was not crucial for the protective functions of canonical inflammasomes in these mice. Taken together, our mouse genetic experiments revealed an essential cooperation between caspase-8 signaling and GSDMD-independent canonical inflammasome signaling to establish intestinal and systemic host defense against gastrointestinal C. rodentium infection.

N-terminal acetylation is a conserved protein modification among eukaryotes. The yeast Saccharomyces cerevisiae is a valuable model system for studying this modification. The bulk of protein N-terminal acetylation in S. cerevisiae is catalyzed by the N-terminal acetyltransferases NatA, NatB, and NatC. Thus far, proteome-wide identification of the in vivo protein sub-strates of yeast NatA and NatB has been performed by N-ter-minomics. Here, we used S. cerevisiae deleted for the NatC catalytic subunit Naa30 and identified 57 yeast NatC substrates by N-terminal combined fractional diagonal chromatography analysis. Interestingly, in addition to the canonical N-termini starting with ML, MI, MF, and MW, yeast NatC substrates also included MY, MK, MM, MA, MV, and MS. However, for some of these substrate types, such as MY, MK, MV, and MS, we also uncovered (residual) non-NatC NAT activity, most likely due to the previously established redundancy between yeast NatC and NatE/Naa50. Thus, we have revealed a complex interplay be-tween different NATs in targeting methionine-starting N -termini in yeast. Furthermore, our results showed that ectopic expression of human NAA30 rescued known NatC phenotypes in naa30 Delta yeast, as well as partially restored the yeast NatC Nt-acetylome. Thus, we demonstrate an evolutionary conservation of NatC from yeast to human thereby underpinning future disease models to study pathogenic NAA30 variants. Overall, this work offers increased biochemical and functional insights into NatC-mediated N-terminal acetylation and provides a basis for future work to pinpoint the specific molecular mechanisms that link the lack of NatC-mediated N-terminal acetylation to phenotypes of NatC deletion yeast.

Mapping protein-protein interactions is crucial to understand protein function. Recent advances in proximity-dependent biotinylation (BioID) coupled to mass spectrometry (MS) allow the characterization of protein complexes in diverse plant models. Here, we describe the use of BioID in hairy root cultures of tomato and provide detailed information on how to analyze the data obtained by MS.

Rhizogenic Agrobacterium strains comprise biotrophic pathogens that cause hairy root disease (HRD) on hydroponically grown Solanaceae and Cucurbitaceae crops, besides being widely explored agents for the creation of hairy root cultures for the sustainable production of plant-specialized metabolites. Hairy root formation is mediated through the expression of genes encoded on the T-DNA of the root-inducing (Ri) plasmid, of which several, including root oncogenic locus B (rolB), play a major role in hairy root development. Despite decades of research, the exact molecular function of the proteins encoded by the rol genes remains enigmatic. Here, by means of TurboID-mediated proximity labeling in tomato (Solanum lycopersicum) hairy roots, we identified the repressor proteins TOPLESS (TPL) and Novel Interactor of JAZ (NINJA) as direct interactors of RolB. Although these interactions allow RolB to act as a transcriptional repressor, our data hint at another in planta function of the RolB oncoprotein. Hence, by a series of plant bioassays, transcriptomic and DNA-binding site enrichment analyses, we conclude that RolB can mitigate the TPL functioning so that it leads to a specific and partial reprogramming of phytohormone signaling, immunity, growth, and developmental processes. Our data support a model in which RolB manipulates host transcription, at least in part, through interaction with TPL, to facilitate hairy root development. Thereby, we provide important mechanistic insights into this renowned oncoprotein in HRD.

Alternative translation initiation is a widespread event in biology that can shape multiple protein forms or proteoforms from a single gene. However, the respective contribution of alternative translation to protein complexity remains largely enigmatic. By complementary ribosome profiling and N-terminal proteomics (i.e., riboproteogenomics), we provide clear-cut evidence for ~90 N-terminal proteoform pairs shaped by (alternative) translation initiation in Arabidopsis thaliana. Next to several cases additionally confirmed by directed mutagenesis, identified alternative protein N-termini follow the enzymatic rules of co-translational N-terminal protein acetylation and initiator methionine removal. In contrast to other eukaryotic models, N-terminal acetylation in plants cannot generally be considered as a proxy of translation initiation because of its posttranslational occurrence on mature proteolytic neo-termini (N-termini) localized in the chloroplast stroma. Quantification of N-terminal acetylation revealed differing co- vs. posttranslational N-terminal acetylation patterns. Intriguingly, our data additionally hints to alternative translation initiation serving as a common mechanism to supply protein copies in multiple cellular compartments, as alternative translation sites are often in close proximity to cleavage sites of N-terminal transit sequences of nuclear-encoded chloroplastic and mitochondrial proteins. Overall, riboproteogenomics screening enables the identification of (differential localized) N-terminal proteoforms raised upon alternative translation.

Genomic studies of bacteria have long pointed toward widespread prevalence of small open reading frames (sORFs) encoding for short proteins, <100 amino acids in length. Despite the mounting genomic evidence of their robust expression, relatively little progress has been made in their mass spectrometry-based detection and various blanket statements have been used to explain this observed discrepancy. In this study, we provide a large-scale riboproteogenomics investigation of the challenging nature of proteomic detection of such small proteins as informed by conditional translation data. A panel of physiochemical properties alongside recently developed mass spectrometry detectability metrics was interrogated to provide a comprehensive evidence-based assessment of sORF-encoded polypeptide (SEP) detectability. Moreover, a large-scale proteomics and translatomics compendium of proteins produced by Salmonella Typhimurium (S. Typhimurium), a model human pathogen, across a panel of growth conditions is presented and used in support of our in silico SEP detectability analysis. This integrative approach is used to provide a data-driven census of small proteins expressed by S. Typhimurium across growth phases and infection-relevant conditions. Taken together, our study pinpoints current limitations in proteomics-based detection of novel small proteins currently missing from bacterial genome annotations.

In the context of host-pathogen interactions, gram-negative bacterial virulence factors, such as effectors, may be transferred from bacterial to eukaryotic host cytoplasm by multicomponent Type III protein secretion systems (T3SSs). Central to Salmonella enterica serovar Typhimurium (S. Typhimurium) pathogenesis is the secretion of over 40 effectors by two T3SSs encoded within pathogenicity islands SPI-1 and SPI-2. These effectors manipulate miscellaneous host cellular processes, such as cytoskeleton organization and immune signaling pathways, thereby permitting host colonization and bacterial dissemination. Recent research on effector biology provided mechanistic insights for some effectors. However, for many effectors, clearly defined roles and host target repertoires—further clarifying effector interconnectivity and virulence networks—are yet to be uncovered. Here we demonstrate the utility of the recently described viral-like particle trapping technology Virotrap as an effective approach to catalog S. Typhimurium effector-host protein complexes (EH-PCs). Mass spectrometry-based Virotrap analysis of the novel E3 ubiquitin ligase SspH2 previously shown to be implicated in modulating actin dynamics and immune signaling, exposed known host interactors PFN1 and−2 besides several putative novel, interconnected host targets. Network analysis revealed an actin (-binding) cluster among the significantly enriched hits for SspH2, consistent with the known localization of the S-palmitoylated effector with actin cytoskeleton components in the host. We show that Virotrap complements the current state-of-the-art toolkit to study protein complexes and represents a valuable means to screen for effector host targets in a high-throughput manner, thereby bridging the knowledge gap between effector-host interplay and pathogenesis.

The evolutionary conserved N-alpha acetyltransferase Naa40p is among the most selective N-terminal acetyltransferases (NATs) identified to date. Here we identified a conserved N-terminally truncated Naa40p proteoform named Naa40p25 or short Naa40p (Naa40S). Intriguingly, although upon ectopic expression in yeast, both Naa40p proteoforms were capable of restoring N-terminal acetylation of the characterized yeast histone H2A Naa40p substrate, the Naa40p histone H4 substrate remained N-terminally free in human haploid cells specifically deleted for canonical Naa40p27 or 237 amino acid long Naa40p (Naa40L), but expressing Naa40S. Interestingly, human Naa40L and Naa40S displayed differential expression and subcellular localization patterns by exhibiting a principal nuclear and cytoplasmic localization, respectively. Furthermore, Naa40L was shown to be N-terminally myristoylated and to interact with N-myristoyltransferase 1 (NMT1), implicating NMT1 in steering Naa40L nuclear import. Differential interactomics data obtained by biotin-dependent proximity labeling (BioID) further hints to context-dependent roles of Naa40p proteoforms. More specifically, with Naa40S representing the main co-translationally acting actor, the interactome of Naa40L was enriched for nucleolar proteins implicated in ribosome biogenesis and the assembly of ribonucleoprotein particles, overall indicating a proteoform-specific segregation of previously reported Naa40p activities. Finally, the yeast histone variant H2A.Z and the transcriptionally regulatory protein Lge1 were identified as novel Naa40p substrates, expanding the restricted substrate repertoire of Naa40p with two additional members and further confirming Lge1 as being the first redundant yNatA and yNatD substrate identified to date.

NatD consist of the evolutionary conserved N-alpha acetyltransferase catalytic subunit Naa40p and is among the most selective N-terminal acetyltransferase (NAT) complexes identified to date. Here we identified a conserved 22 amino acid N-terminally truncated Naa40 proteoform named Naa40p25 or short Naa40 (Naa40 S ). Intriguingly, while upon ectopic expression in yeast, both Naa40p proteoforms were capable of restoring N-terminal acetylation of the well characterized yeast histone H2A Naa40p substrate, the Naa40p histone H4 substrate remained N-terminally free in human haploid cells specifically deleted for canonical Naa40p27 or 237 amino acid long Naa40 (Naa40 L ), but expressing Naa40 S . Interestingly, human Naa40 L and Naa40 S displayed differential expression and subcellular localization patterns by exhibiting a principal nuclear and cytoplasmic localization, respectively. Further, since Naa40 L was identified as being N-terminally myristoylated and shown to interact with N-myristoyltransferase 1 (NMT1), NMT1 may steer Naa40 L nuclear import. Differential interactomics data obtained by biotin-dependent proximity labeling (BioID) further hints to context dependent roles of Naa40p proteoforms. More specifically, with Naa40 S representing the main co-translationally acting actor, the interactome of Naa40 L was enriched for nucleolar proteins implicated in ribosome biogenesis and the assembly of ribonucleoprotein particles, overall indicating a proteoform-specific segregation of previously reported Naa40p activities. Finally, the yeast histone variant H2A.Z and the transcriptionally regulatory protein Lge1 were identified as novel Naa40p substrates, expanding the restricted substrate repertoire of Naa40p with two additional members and further confirming Lge1 as being the first redundant yNatA and yNatD substrate identified to date.

Excision of the N-terminal initiator methionine (iMet) residue from nascent peptide chains is an essential and omnipresent protein modification carried out by methionine aminopeptidases (MetAPs) that accounts for a major source of N-terminal proteoform diversity. Although MetAP2 is known to be implicated in processes such as angiogenesis and proliferation in mammals, the physiological role of MetAP1 is much less clear. In this report we studied the omics-wide effects of human MetAP1 deletion and general MetAP inhibition. The levels of iMet retention are inversely correlated with cellular proliferation rates. Further, despite the increased MetAP2 expression on MetAP1 deletion, MetAP2 was unable to restore processing of Met-Ser-, Met-Pro-, and Met-Ala- starting N termini as inferred from the iMet retention profiles observed, indicating a higher activity of MetAP1 over these N termini. Proteome and transcriptome expression profiling point to differential expression of proteins implicated in lipid metabolism, cytoskeleton organization, cell proliferation and protein synthesis upon perturbation of MetAP activity.