An N-end rule pathway that recognizes proline and destroys gluconeogenic enzymes.

Cells synthesize glucose if deprived of it, and destroy gluconeogenic enzymes upon return to glucose-replete conditions. We found that the Gid4 subunit of the ubiquitin ligase GID in the yeast Saccharomyces cerevisiae targeted the gluconeogenic enzymes Fbp1, Icl1, and Mdh2 for degradation. Gid4 recognized the N-terminal proline (Pro) residue and the ~5-residue-long adjacent sequence motifs. Pck1, the fourth gluconeogenic enzyme, contains Pro at position 2; Gid4 directly or indirectly recognized Pro at position 2 of Pck1, contributing to its targeting. These and related results identified Gid4 as the recognition component of the GID-based proteolytic system termed the Pro/N-end rule pathway. Substrates of this pathway include gluconeogenic enzymes that bear either the N-terminal Pro residue or a Pro at position 2, together with adjacent sequence motifs.

Analyzing N-terminal Arginylation through the Use of Peptide Arrays and Degradation Assays.

Nα-terminal arginylation (Nt-arginylation) of proteins is mediated by the Ate1 arginyltransferase (R-transferase), a component of the Arg/N-end rule pathway. This proteolytic system recognizes proteins containing N-terminal degradation signals called N-degrons, polyubiquitylates these proteins, and thereby causes their degradation by the proteasome. The definitively identified ("canonical") residues that are Nt-arginylated by R-transferase are N-terminal Asp, Glu, and (oxidized) Cys. Over the last decade, several publications have suggested (i) that Ate1 can also arginylate non-canonical N-terminal residues; (ii) that Ate1 is capable of arginylating not only α-amino groups of N-terminal residues but also γ-carboxyl groups of internal (non-N-terminal) Asp and Glu; and (iii) that some isoforms of Ate1 are specific for substrates bearing N-terminal Cys residues. In the present study, we employed arrays of immobilized 11-residue peptides and pulse-chase assays to examine the substrate specificity of mouse R-transferase. We show that amino acid sequences immediately downstream of a substrate's canonical (Nt-arginylatable) N-terminal residue, particularly a residue at position 2, can affect the rate of Nt-arginylation by R-transferase and thereby the rate of degradation of a substrate protein. We also show that the four major isoforms of mouse R-transferase have similar Nt-arginylation specificities in vitro, contrary to the claim about the specificity of some Ate1 isoforms for N-terminal Cys. In addition, we found no evidence for a significant activity of the Ate1 R-transferase toward previously invoked non-canonical N-terminal or internal amino acid residues. Together, our results raise technical concerns about earlier studies that invoked non-canonical arginylation specificities of Ate1.

Degradation of Serotonin N-Acetyltransferase, a Circadian Regulator, by the N-end Rule Pathway.

Serotonin N-acetyltransferase (AANAT) converts serotonin to N-acetylserotonin (NAS), a distinct biological regulator and the immediate precursor of melatonin, a circulating hormone that influences circadian processes, including sleep. N-terminal sequences of AANAT enzymes vary among vertebrates. Mechanisms that regulate the levels of AANAT are incompletely understood. Previous findings were consistent with the possibility that AANAT may be controlled through its degradation by the N-end rule pathway. By expressing the rat and human AANATs and their mutants not only in mammalian cells but also in the yeast Saccharomyces cerevisiae, and by taking advantage of yeast genetics, we show here that two "complementary" forms of rat AANAT are targeted for degradation by two "complementary" branches of the N-end rule pathway. Specifically, the N(α)-terminally acetylated (Nt-acetylated) Ac-AANAT is destroyed through the recognition of its Nt-acetylated N-terminal Met residue by the Ac/N-end rule pathway, whereas the non-Nt-acetylated AANAT is targeted by the Arg/N-end rule pathway, which recognizes the unacetylated N-terminal Met-Leu sequence of rat AANAT. We also show, by constructing lysine-to-arginine mutants of rat AANAT, that its degradation is mediated by polyubiquitylation of its Lys residue(s). Human AANAT, whose N-terminal sequence differs from that of rodent AANATs, is longer-lived than its rat counterpart and appears to be refractory to degradation by the N-end rule pathway. Together, these and related results indicate both a major involvement of the N-end rule pathway in the control of rodent AANATs and substantial differences in the regulation of rodent and human AANATs that stem from differences in their N-terminal sequences.

Degradation of the Separase-cleaved Rec8, a Meiotic Cohesin Subunit, by the N-end Rule Pathway.

The Ate1 arginyltransferase (R-transferase) is a component of the N-end rule pathway, which recognizes proteins containing N-terminal degradation signals called N-degrons, polyubiquitylates these proteins, and thereby causes their degradation by the proteasome. Ate1 arginylates N-terminal Asp, Glu, or (oxidized) Cys. The resulting N-terminal Arg is recognized by ubiquitin ligases of the N-end rule pathway. In the yeastSaccharomyces cerevisiae, the separase-mediated cleavage of the Scc1/Rad21/Mcd1 cohesin subunit generates a C-terminal fragment that bears N-terminal Arg and is destroyed by the N-end rule pathway without a requirement for arginylation. In contrast, the separase-mediated cleavage of Rec8, the mammalian meiotic cohesin subunit, yields a fragment bearing N-terminal Glu, a substrate of the Ate1 R-transferase. Here we constructed and used a germ cell-confinedAte1(-/-)mouse strain to analyze the separase-generated C-terminal fragment of Rec8. We show that this fragment is a short-lived N-end rule substrate, that its degradation requires N-terminal arginylation, and that maleAte1(-/-)mice are nearly infertile, due to massive apoptotic death ofAte1(-/-)spermatocytes during the metaphase of meiosis I. These effects ofAte1ablation are inferred to be caused, at least in part, by the failure to destroy the C-terminal fragment of Rec8 in the absence of N-terminal arginylation.

Control of mammalian G protein signaling by N-terminal acetylation and the N-end rule pathway.

Rgs2, a regulator of G proteins, lowers blood pressure by decreasing signaling through Gαq. Human patients expressing Met-Leu-Rgs2 (ML-Rgs2) or Met-Arg-Rgs2 (MR-Rgs2) are hypertensive relative to people expressing wild-type Met-Gln-Rgs2 (MQ-Rgs2). We found that wild-type MQ-Rgs2 and its mutant, MR-Rgs2, were destroyed by the Ac/N-end rule pathway, which recognizes N(α)-terminally acetylated (Nt-acetylated) proteins. The shortest-lived mutant, ML-Rgs2, was targeted by both the Ac/N-end rule and Arg/N-end rule pathways. The latter pathway recognizes unacetylated N-terminal residues. Thus, the Nt-acetylated Ac-MX-Rgs2 (X = Arg, Gln, Leu) proteins are specific substrates of the mammalian Ac/N-end rule pathway. Furthermore, the Ac/N-degron of Ac-MQ-Rgs2 was conditional, and Teb4, an endoplasmic reticulum (ER) membrane-embedded ubiquitin ligase, was able to regulate G protein signaling by targeting Ac-MX-Rgs2 proteins for degradation through their N(α)-terminal acetyl group.

Liat1, an arginyltransferase-binding protein whose evolution among primates involved changes in the numbers of its 10-residue repeats.

The arginyltransferase Ate1 is a component of the N-end rule pathway, which recognizes proteins containing N-terminal degradation signals called N-degrons, polyubiquitylates these proteins, and thereby causes their degradation by the proteasome. At least six isoforms of mouse Ate1 are produced through alternative splicing of Ate1 pre-mRNA. We identified a previously uncharacterized mouse protein, termed Liat1 (ligand of Ate1), that interacts with Ate1 but does not appear to be its arginylation substrate. Liat1 has a higher affinity for the isoforms Ate1(1A7A) and Ate1(1B7A). Liat1 stimulated the in vitro N-terminal arginylation of a model substrate by Ate1. All examined vertebrate and some invertebrate genomes encode proteins sequelogous (similar in sequence) to mouse Liat1. Sequelogs of Liat1 share a highly conserved ∼30-residue region that is shown here to be required for the binding of Liat1 to Ate1. We also identified non-Ate1 proteins that interact with Liat1. In contrast to Liat1 genes of nonprimate mammals, Liat1 genes of primates are subtelomeric, a location that tends to confer evolutionary instability on a gene. Remarkably, Liat1 proteins of some primates, from macaques to humans, contain tandem repeats of a 10-residue sequence, whereas Liat1 proteins of other mammals contain a single copy of this motif. Quantities of these repeats are, in general, different in Liat1 of different primates. For example, there are 1, 4, 13, 13, 17, and 17 repeats in the gibbon, gorilla, orangutan, bonobo, neanderthal, and human Liat1, respectively, suggesting that repeat number changes in this previously uncharacterized protein may contribute to evolution of primates.

Gamma-aminobutyric acid and glutamate differentially regulate intracellular calcium concentrations in mouse gonadotropin-releasing hormone neurons.

Multiple factors regulate the activity of the GnRH neurons responsible for controlling fertility. Foremost among neuronal inputs to GnRH neurons are those using the amino acids glutamate and gamma-aminobutyric acid (GABA). The present study used a GnRH-Pericam transgenic mouse line, enabling live cell imaging of intracellular calcium concentrations ([Ca(2+)](i)) to evaluate the effects of glutamate and GABA signaling on [Ca(2+)](i) in peripubertal and adult mouse GnRH neurons. Activation of GABA(A), N-methyl-d-aspartate, or alpha-amino-3-hydroxyl-5-methyl-4-isoxazole propionate acid (AMPA) receptors was found to evoke an increase in [Ca(2+)](i), in subpopulations of GnRH neurons. Approximately 70% of GnRH neurons responded to GABA, regardless of postnatal age or sex. Many fewer (approximately 20%) GnRH neurons responded to N-methyl-d-aspartate, and this was not influenced by postnatal age or sex. In contrast, about 65% of adult male and female GnRH neurons responded to AMPA compared with about 14% of male and female peripubertal mice (P < 0.05). The mechanisms underlying the ability of GABA and AMPA to increase [Ca(2+)](i) in adult GnRH neurons were evaluated pharmacologically. Both GABA and AMPA were found to evoke [Ca(2+)](i) increases through a calcium-induced calcium release mechanism involving internal calcium stores and inositol-1,4,5-trisphosphate receptors. For GABA, the initial increase in [Ca(2+)](i) originated from GABA(A) receptor-mediated activation of L-type voltage-gated calcium channels, whereas for AMPA this appeared to involve direct calcium entry through the AMPA receptor. These observations show that all of the principal amino acid receptors are able to control [Ca(2+)](i) in GnRH neurons but that they do so in a postnatal age- and intracellular pathway-specific manner.

The gonadotropin-releasing hormone (GnRH) neuronal population is normal in size and distribution in GnRH-deficient and GnRH receptor-mutant hypogonadal mice.

Hypothalamic GnRH neurons are essential for initiation and regulation of reproductive function. In addition to pituitary gonadotrope stimulation, activity of GnRH through its receptor (GnRHR) has been suggested to include autocrine regulation of the GnRH neuron. Two hypogonadal mouse strains, the Gnrh1 mutant (hpg) mice and Gnrhr mutant mice were used to investigate the potential role of GnRH signaling in the proper development and maintenance of GnRH neurons. Immunocytochemical analysis of heterozygous hpg mice revealed a GnRH neuron population that was normal in size and distribution, indicating no effect from reduced Gnrh1 gene dosage on the neurons themselves. To visualize GnRH neurons in homozygous GnRH-deficient hpg mice, heterozygous hpg mice were crossed with GnRH-green fluorescent protein (GFP) transgenic mice with targeted expression of the GFP reporter gene in GnRH neurons. Analysis of forebrains of homozygous hpg/GFP-positive mice immunostained for GFP revealed a normal population size and appropriate distribution of GnRH neurons in hpg mice, with immunoreactive neuronal processes present at the median eminence. Similarly, adult mice deficient in functional GnRHR possessed a full complement of GnRH neurons in the basal forebrain that was indistinguishable from the distribution of GnRH neurons in their wild-type counterparts. Moreover, hpg/GFP neurons retained the ability to generate spontaneous bursts of action potential firing activity, suggesting that GnRH peptide is not required for this function. These data establish that autocrine-paracrine GnRH-signaling is not a prerequisite for the developmental migration of GnRH neurons into the brain or for the projection of GnRH neurosecretory axons.