The Final Maturation State of ß-actin Involves N-terminal Acetylation by NAA80, not N-terminal Arginylation by ATE1.

Actin is a hallmark protein of the cytoskeleton in eukaryotic cells, affecting a range of cellular functions. Actin dynamics is regulated through a myriad of actin-binding proteins and post-translational modifications. The mammalian actin family consists of six different isoforms, which vary slightly in their N-terminal (Nt) sequences. During and after synthesis, actins undergo an intricate Nt-processing that yields mature actin isoforms. The ubiquitously expressed cytoplasmic ß-actin is Nt-acetylated by N-alpha acetyltransferase 80 (NAA80) yielding the Nt-sequence Ac-DDDI-. In addition, ß-actin was also reported to be Nt-arginylated by arginyltransferase 1 (ATE1) after further peptidase-mediated processing, yielding RDDI-. To characterize in detail the Nt-processing of actin, we used state-of-the-art proteomics. To estimate the relative cellular levels of Nt-modified proteoforms of actin, we employed NAA80-lacking cells, in which actin was not Nt-acetylated. We found that targeted proteomics is superior to a commercially available antibody previously used to analyze Nt-arginylation of ß-actin. Significantly, despite the use of sensitive mass spectrometry-based techniques, we could not confirm the existence of the previously claimed Nt-arginylated ß-actin (RDDI-) in either wildtype or NAA80-lacking cells. A very minor level of Nt-arginylation of the initially cleaved ß-actin (DDDI-) could be identified, but only in NAA80-lacking cells, not in wildtype cells. We also identified small fractions of cleaved and unmodified ß-actin (DDI-) as well as cleaved and Nt-acetylated ß-actin (Ac-DDI-). In sum, we show that the multi-step Nt-maturation of ß-actin is terminated by NAA80, which Nt-acetylates the exposed Nt-Asp residues, in the virtual absence of previously claimed Nt-arginylation.

Actin polymerization and cell motility are affected by NAA80-mediated posttranslational N-terminal acetylation of actin.

Actin is the most abundant protein in our cells, and also one of the most studied. Nevertheless, an important modifier of actin, the N-terminal acetyltransferase (NAT) for actin, remained unknown until now. The recent identification of the enzyme that catalyzes actin acetylation, has opened up for functional studies of unacetylated actin using knockout cells. This enzyme, called NAA80 (Nα-acetyltransferase 80) or NatH, belongs to the NAT family of enzymes, which together provides N-terminal acetylation for around 80 % of the human proteome. In many cases, N-terminal acetylation is essential. In the case of actin, the acetyl group that NAA80 attaches to actin plays an important role in actin's polymerization properties as well as in actin's function in cell migration.

Intermediate compartment (IC): from pre-Golgi vacuoles to a semi-autonomous membrane system.

Despite its discovery more than three decades ago and well-established role in protein sorting and trafficking in the early secretory pathway, the intermediate compartment (IC) has remained enigmatic. The prevailing view is that the IC evolved as a specialized organelle to mediate long-distance endoplasmic reticulum (ER)-Golgi communication in metazoan cells, but is lacking in other eukaryotes, such as plants and fungi. However, this distinction is difficult to reconcile with the high conservation of the core machineries that regulate early secretory trafficking from yeast to man. Also, it has remained unclear whether the pleiomorphic IC components-vacuoles, tubules and vesicles-represent transient transport carriers or building blocks of a permanent pre-Golgi organelle. Interestingly, recent studies have revealed that the IC maintains its compositional, structural and spatial properties throughout the cell cycle, supporting a model that combines the dynamic and stable aspects of the organelle. Moreover, the IC has been assigned novel functions, such as cell signaling, Golgi-independent trafficking and autophagy. The emerging permanent nature of the IC and its connections with the centrosome and the endocytic recycling system encourage reconsideration of its relationship with the Golgi ribbon, role in Golgi biogenesis and ubiquitous presence in eukaryotic cells.

NAA80 is actin's N-terminal acetyltransferase and regulates cytoskeleton assembly and cell motility.

Actin, one of the most abundant proteins in nature, participates in countless cellular functions ranging from organelle trafficking and pathogen motility to cell migration and regulation of gene transcription. Actin's cellular activities depend on the dynamic transition between its monomeric and filamentous forms, a process exquisitely regulated in cells by a large number of actin-binding and signaling proteins. Additionally, several posttranslational modifications control the cellular functions of actin, including most notably N-terminal (Nt)-acetylation, a prevalent modification throughout the animal kingdom. However, the biological role and mechanism of actin Nt-acetylation are poorly understood, and the identity of actin's N-terminal acetyltransferase (NAT) has remained a mystery. Here, we reveal that NAA80, a suggested NAT enzyme whose substrate specificity had not been characterized, is Nt-acetylating actin. We further show that actin Nt-acetylation plays crucial roles in cytoskeletal assembly in vitro and in cells. The absence of Nt-acetylation leads to significant differences in the rates of actin filament depolymerization and elongation, including elongation driven by formins, whereas filament nucleation by the Arp2/3 complex is mostly unaffected. NAA80-knockout cells display severely altered cytoskeletal organization, including an increase in the ratio of filamentous to globular actin, increased filopodia and lamellipodia formation, and accelerated cell motility. Together, the results demonstrate NAA80's role as actin's NAT and reveal a crucial role for actin Nt-acetylation in the control of cytoskeleton structure and dynamics.

Phosphorylation at serine 31 targets tyrosine hydroxylase to vesicles for transport along microtubules.

Tyrosine hydroxylase (TH) catalyzes the conversion of l-tyrosine into l-DOPA, which is the rate-limiting step in the synthesis of catecholamines, such as dopamine, in dopaminergergic neurons. Low dopamine levels and death of the dopaminergic neurons are hallmarks of Parkinson's disease (PD), where α-synuclein is also a key player. TH is highly regulated, notably by phosphorylation of several Ser/Thr residues in the N-terminal tail. However, the functional role of TH phosphorylation at the Ser-31 site (THSer(P)-31) remains unclear. Here, we report that THSer(P)-31 co-distributes with the Golgi complex and synaptic-like vesicles in rat and human dopaminergic cells. We also found that the TH microsomal fraction content decreases after inhibition of cyclin-dependent kinase 5 (Cdk5) and ERK1/2. The cellular distribution of an overexpressed phospho-null mutant, TH1-S31A, was restricted to the soma of neuroblastoma cells, with decreased association with the microsomal fraction, whereas a phospho-mimic mutant, TH1-S31E, was distributed throughout the soma and neurites. TH1-S31E associated with vesicular monoamine transporter 2 (VMAT2) and α-synuclein in neuroblastoma cells, and endogenous THSer(P)-31 was detected in VMAT2- and α-synuclein-immunoprecipitated mouse brain samples. Microtubule disruption or co-transfection with α-synuclein A53T, a PD-associated mutation, caused TH1-S31E accumulation in the cell soma. Our results indicate that Ser-31 phosphorylation may regulate TH subcellular localization by enabling its transport along microtubules, notably toward the projection terminals. These findings disclose a new mechanism of TH regulation by phosphorylation and reveal its interaction with key players in PD, opening up new research avenues for better understanding dopamine synthesis in physiological and pathological states.

First Things First: Vital Protein Marks by N-Terminal Acetyltransferases.

N-terminal (Nt) acetylation is known to be a highly abundant co-translational protein modification, but the recent discovery of Golgi- and chloroplast-resident N-terminal acetyltransferases (NATs) revealed that it can also be added post-translationally. Nt-acetylation may act as a degradation signal in a novel branch of the N-end rule pathway, whose functions include the regulation of human blood pressure. Nt-acetylation also modulates protein interactions, targeting, and folding. In plants, Nt-acetylation plays a role in the control of resistance to drought and in regulation of immune responses. Mutations of specific human NATs that decrease their activity can cause either the lethal Ogden syndrome or severe intellectual disability and cardiovascular defects. In sum, recent advances highlight Nt-acetylation as a key factor in many biological pathways.

Microscopy-based Saccharomyces cerevisiae complementation model reveals functional conservation and redundancy of N-terminal acetyltransferases.

N-terminal acetylation is a highly abundant protein modification catalyzed by N-terminal acetyltransferases (NATs) NatA-NatG. The Saccharomyces cerevisiae protein Arl3 depends on interaction with Sys1 for its localization to the Golgi and this targeting strictly requires NatC-mediated N-terminal acetylation of Arl3. We utilized the Arl3 acetylation-dependent localization phenotype as a model system for assessing the functional conservation and in vivo redundancy of several human NATs. The catalytic subunit of human NatC, hNaa30 (Mak3), restored Arl3 localization in the absence of yNaa30, but only in the presence of either yeast or human Naa35 subunit (Mak10). In contrast, hNaa35 was not able to replace its yeast orthologue without the co-expression of hNaa30, suggesting co-evolution of the two NatC subunits. The most recently discovered and organellar human NAT, NatF/Naa60, restored the Golgi localization of Arl3 in the absence of yNaa30. Interestingly, this was also true for hNaa60 lacking its membrane-binding domain whereas hNaa50 did not complement NatC function. This in vivo redundancy reflects NatC and NatF´s overlapping in vitro substrate specificities. The yeast model presented here provides a robust and rapid readout of NatC and NatF activity in vivo, and revealed evolutionary conservation of the NatC complex and redundancy between NatC and NatF.

An organellar nα-acetyltransferase, naa60, acetylates cytosolic N termini of transmembrane proteins and maintains Golgi integrity.

N-terminal acetylation is a major and vital protein modification catalyzed by N-terminal acetyltransferases (NATs). NatF, or Nα-acetyltransferase 60 (Naa60), was recently identified as a NAT in multicellular eukaryotes. Here, we find that Naa60 differs from all other known NATs by its Golgi localization. A new membrane topology assay named PROMPT and a selective membrane permeabilization assay established that Naa60 faces the cytosolic side of intracellular membranes. An Nt-acetylome analysis of NAA60-knockdown cells revealed that Naa60, as opposed to other NATs, specifically acetylates transmembrane proteins and has a preference for N termini facing the cytosol. Moreover, NAA60 knockdown causes Golgi fragmentation, indicating an important role in the maintenance of the Golgi's structural integrity. This work identifies a NAT associated with membranous compartments and establishes N-terminal acetylation as a common modification among transmembrane proteins, a thus-far poorly characterized part of the N-terminal acetylome.

Endocytosis of secreted carboxyl ester lipase in a syndrome of diabetes and pancreatic exocrine dysfunction.

Maturity-onset diabetes of the young, type 8 (MODY8) is characterized by a syndrome of autosomal dominantly inherited diabetes and exocrine pancreatic dysfunction. It is caused by deletion mutations in the last exon of the carboxyl ester lipase (CEL) gene, resulting in a CEL protein with increased tendency to aggregate. In this study we investigated the intracellular distribution of the wild type (WT) and mutant (MUT) CEL proteins in cellular models. We found that both CEL-WT and CEL-MUT were secreted via the endoplasmic reticulum and Golgi compartments. However, their subcellular distributions differed, as only CEL-MUT was observed as an aggregate at the cell surface and inside large cytoplasmic vacuoles. Many of the vacuoles were identified as components of the endosomal system, and after its secretion, the mutant CEL protein was re-internalized, transported to the lysosomes, and degraded. Internalization of CEL-MUT also led to reduced viability of pancreatic acinar and beta cells. These findings may have implications for the understanding of how the acinar-specific CEL-MUT protein causes both exocrine and endocrine pancreatic disease.

Division of the intermediate compartment at the onset of mitosis provides a mechanism for Golgi inheritance.

As mammalian cells prepare for mitosis, the Golgi ribbon is first unlinked into its constituent stacks and then transformed into spindle-associated, pleiomorphic membrane clusters in a process that remains enigmatic. Also, it remains unclear whether Golgi inheritance involves the incorporation of Golgi enzymes into a pool of coat protein I (COPI) vesicles, or their COPI-independent transfer to the endoplasmic reticulum (ER). Based on the observation that the intermediate compartment (IC) at the ER-Golgi boundary is connected to the centrosome, we examined its mitotic fate and possible role in Golgi breakdown. The use of multiple imaging techniques and markers revealed that the IC elements persist during the M phase, maintain their compositional and structural properties and remain associated with the mitotic spindle, forming circular arrays at the spindle poles. At G2/M transition, the movement of the pericentrosomal domain of the IC (pcIC) to the cell centre and its expansion coincide with the unlinking of the Golgi ribbon. At prophase, coupled to centrosome separation, the pcIC divides together with recycling endosomes, providing novel landmarks for mitotic entry. We provide evidence that the permanent IC elements function as way stations during the COPI-dependent dispersal of Golgi components at prometa- and metaphase, indicating that they correspond to the previously described Golgi clusters. In addition, they continue to communicate with the vesicular 'Golgi haze' and thus are likely to provide templates for Golgi reassembly. These results implicate the IC in mitotic Golgi inheritance, resulting in a model that integrates key features of the two previously proposed pathways.

Emerging new roles of the pre-Golgi intermediate compartment in biosynthetic-secretory trafficking.

The intermediate compartment (IC) between the endoplasmic reticulum (ER) and the Golgi apparatus appears to constitute an autonomous organelle composed of spatially and functionally distinct, but interconnected, vacuolar and tubular subdomains. In mammalian cells the IC network is stably anchored at the cell center, communicating directly with the endocytic pathway via a pericentrosomal membrane system (PCMS). This finding suggests that the secretory pathway divides at the level of the IC, which functions as a sorting station both in Golgi-dependent and -independent trafficking. The tubular subdomain of the IC is capable of expansion in accordance with its proposed biosynthetic functions such as cholesterol synthesis.

The function of the intermediate compartment in pre-Golgi trafficking involves its stable connection with the centrosome.

Because the functional borders of the intermediate compartment (IC) are not well defined, the spatial map of the transport machineries operating between the endoplasmic reticulum (ER) and the Golgi apparatus remains incomplete. Our previous studies showed that the IC consists of interconnected vacuolar and tubular parts with specific roles in pre-Golgi trafficking. Here, using live cell imaging, we demonstrate that the tubules containing the GTPase Rab1A create a long-lived membrane compartment around the centrosome. Separation of this pericentrosomal domain of the IC from the Golgi ribbon, due to centrosome motility, revealed that it contains a distinct pool of COPI coats and acts as a temperature-sensitive way station in post-ER trafficking. However, unlike the Golgi, the pericentrosomal IC resists the disassembly of COPI coats by brefeldin A, maintaining its juxtaposition with the endocytic recycling compartment, and operation as the focal point of a dynamic tubular network that extends to the cell periphery. These results provide novel insight into the compartmental organization of the secretory pathway and Golgi biogenesis. Moreover, they reveal a direct functional connection between the IC and the endosomal system, which evidently contributes to unconventional transport of the cystic fibrosis transmembrane conductance regulator to the cell surface.

Use of polarized PC12 cells to monitor protein localization in the early biosynthetic pathway.

A prerequisite for understanding the cellular functions of an unknown protein is the establishment of its subcellular localization. As increasing numbers of novel proteins of the biosynthetic pathway are currently being identified, accessible new methods are required to facilitate their localization. Differentiating rat pheochromocytoma (PC12) cells reorganize their biosynthetic membrane compartments as they develop neurite-like processes. The authors recently showed that polarization of these cells involves the expansion of the intermediate compartment (IC) between the rough endoplasmic reticulum (RER) and the Golgi apparatus. Tubules emerging from the vacuolar parts of the IC move to the developing neurites accumulating in their growth cones, whereas the vacuoles, like RER and Golgi, remain in the cell body. Thus, polarized PC12 cells enhance the resolution for immunofluorescence microscopic mapping of protein localization in the early biosynthetic pathway. The authors also describe here a rapid cell fractionation protocol employing velocity sedimentation in iodixanol gradients that allows one-step separation of the pre-Golgi vacuoles, tubules, and RER.

Rab1 defines a novel pathway connecting the pre-Golgi intermediate compartment with the cell periphery.

The function of the pre-Golgi intermediate compartment (IC) and its relationship with the endoplasmic reticulum (ER) and Golgi remain only partially understood. Here, we report striking segregation of IC domains in polarized PC12 cells that develop neurite-like processes. Differentiation involves expansion of the IC and movement of Rab1-containing tubules to the growth cones of the neurites, whereas p58- and COPI-positive IC elements, like rough ER and Golgi, remain in the cell body. Exclusion of Rab1 effectors p115 and GM130 from the neurites further indicated that the centrifugal, Rab1-mediated pathway has functions that are not directly related to ER-to-Golgi trafficking. Disassembly of COPI coats did not affect this pathway but resulted in missorting of p58 to the neurites. Live cell imaging showed that green fluorescent protein (GFP)-Rab1A-containing IC elements move bidirectionally both within the neurites and cell bodies, interconnecting different ER exit sites and the cis-Golgi region. Moreover, in nonpolarized cells GFP-Rab1A-positive tubules moved centrifugally towards the cell cortex. Hydroxymethylglutaryl-CoA reductase, the key enzyme of cholesterol biosynthesis, colocalized with slowly sedimenting, Rab1-enriched membranes when the IC subdomains were separated by velocity sedimentation. These results reveal a novel pathway directly connecting the IC with the cell periphery and suggest that this Rab1-mediated pathway is linked to the dynamics of smooth ER.