Alternative splicing results in differential expression, activity, and localization of the two forms of arginyl-tRNA-protein transferase, a component of the N-end rule pathway

Abstract

The N-end rule relates the in vivo half-life of a protein to the identity of its N-terminal residue. The underlying ubiquitin-dependent proteolytic system, called the N-end rule pathway, is organized hierarchically: N-terminal aspartate and glutamate (and also cysteine in metazoans) are secondary destabilizing residues, in that they function through their conjugation, by arginyl-tRNA-protein transferase (R-transferase), to arginine, a primary destabilizing residue. We isolated cDNA encoding the 516-residue mouse R-transferase, ATE1p, and found two species, termed Ate1-1 and Ate1-2. The Ate1 mRNAs are produced through a most unusual alternative splicing that retains one or the other of the two homologous 129-bp exons, which are adjacent in the mouse Ate1 gene. Human ATE1 also contains the alternative 129-bp exons, whereas the plant (Arabidopsis thaliana) and fly (Drosophila melanogaster) Ate1 genes encode a single form of ATE1p. A fusion of ATE1-1p with green fluorescent protein (GFP) is present in both the nucleus and the cytosol, whereas ATE1-2p-GFP is exclusively cytosolic. Mouse ATE1-1p and ATE1-2p were examined by expressing them in ate1Delta Saccharomyces cerevisiae in the presence of test substrates that included Asp-betagal (beta-galactosidase) and Cys-betagal. Both forms of the mouse R-transferase conferred instability on Asp-betagal (but not on Cys-betagal) through the arginylation of its N-terminal Asp, the ATE1-1p enzyme being more active than ATE1-2p. The ratio of Ate1-1 to Ate1-2 mRNA varies greatly among the mouse tissues; it is approximately 0.1 in the skeletal muscle, approximately 0.25 in the spleen, approximately 3.3 in the liver and brain, and approximately 10 in the testis, suggesting that the two R-transferases are functionally distinct.

FIG. 1

Comparison of enzymatic reactions that underlie the activity of the tertiary and secondary destabilizing residues among eukaryotes. (A) Mammals (reference and this work); (B) the yeast S. cerevisiae (5). N-terminal residues are indicated by single-letter abbreviations for amino acids; ovals denote the rest of a protein substrate. The Ntan1-encoded mammalian Nt-amidase converts N-terminal Asn to Asp, whereas N-terminal Gln is deamidated by a distinct Nt-amidase that remains to be identified (19, 51). In contrast, the yeast Nt-amidase can deamidate either N-terminal Asn or Gln (6). The secondary destabilizing residues Asp and Glu are arginylated by the mammalian ATE1-1p or ATE1-2p R-transferase (see Results). A Cys-specific mammalian R-transferase remains to be identified (see Results). N-terminal Arg, one of the primary destabilizing residues (54), is recognized by N-recognin (E3) (see the introduction).

FIG. 2

Two forms of the mouse Ate1 cDNA and the ATE protein family. (A) The mouse Ate1-1 and Ate1-2 cDNAs and their products. The nucleotide sequences of Ate1-2 identical to those of Ate1-1 (everywhere except for the 129-bp region) are indicated by dashes. In the region of the alternative 129-bp exons of Ate1-1 and Ate1-2, white-on-black and gray shadings highlight, respectively, identical and similar residues. The circled Cys residues are homologous to those that are important for the enzymatic activity of S. cerevisiae Ate1p (32). (B) The ATE protein family and the origins of the alternative 129-bp exons. Alignment of the sequences of mouse ATE1-1p (Mm-Ate1-1), A. thaliana Ate1p (At-Ate1), D. melanogaster Ate1p (Dm-Ate1), C. elegans Ate1p (Ce-Ate1), and S. cerevisiae Ate1p (Sc-Ate1) (accession no. J05404). Similar residues (gray) were grouped as follows: M, L, I, and V; D, E, N, and Q; R, K, and H; Y, F, and W; S, A, and T. The region encoded by the alternative 129-bp exons of mouse Ate1 is highlighted by a thick line. Of the Cys residues that are conserved among all ATE proteins, the ones required and not required for the enzymatic activity of S. cerevisiae Ate1p (32) are indicated, respectively, by ▾ and ▿. The N-terminally truncated mouse ATE1-1p and ATE1-2p proteins which began at Met-42 (•) lacked the R-transferase activity (data not shown). The highly variable C-terminal regions of ATE proteins were omitted from the alignment. The sequences were aligned using PileUp program (Wisconsin Package; Genetics Computer Group, Madison, Wis.). Gaps (−) were introduced to optimize the alignment. The residue numbers are on the right of the sequences. The sequence of C. elegans Ate1p appears to lack the N-terminal region of other ATE proteins because of an error in defining the Ate1 ORF in the genomic DNA sequence (accession no. Z21146). (C) Alignment of the 43-residue regions that are encoded by the alternative 129-bp exons in mammalian Ate1. Sequences shown: mouse (Mm-Ate1-1 and Mm-Ate1-2), human (Hs-Ate1-1 and Hs-Ate1-2), D. melanogaster (Dm-Ate1), C. elegans (Ce-Ate1), A. thaliana (At-Ate1), S. pombe (Sp-Ate1; accession no. Z99568), and S. cerevisiae (Sc-Ate1) (accession no. J05404). The degrees of identity and similarity of ATE proteins to the deduced amino acid sequences of the M8 (mouse ATE1-1p) or M3 (mouse ATE1-2p) exon of the mouse Ate1 gene are indicated on the right. The residues conserved among all of the compared sequences are indicated above the alignment.

FIG. 3

Expression of Ate1-1 and Ate1-2 mRNAs in different mouse tissues and mouse embryonic fibroblasts (MEF). Two forms of Ate1 cDNA (Fig. 2A) were amplified in a single reaction by RT-PCR, using the same primers, to yield 624-bp fragments that included the region of the alternative 129-bp exons (see Materials and Methods). The 624-bp fragments were digested with ScrFI, which produced a 231-bp fragment (a mixture of Ate1-1 and Ate1-2), a 284-bp, Ate1-1-specific fragment, and a 317-bp, Ate1-2-specific fragment. The untreated (lanes a) and ScrFI-treated (lanes b) samples from different mouse tissues were analyzed by electrophoresis in a 2% agarose gel. The ratio of the two forms of Ate1 mRNA, defined as the ratio of the 284-bp (Ate1-1) fragment to the 317-bp (Ate1-2) fragment, was determined by analyzing serially diluted samples and comparing the resulting band intensities (data not shown). sk., skeletal.

FIG. 4

The two forms of mouse Ate1 mRNA are produced by alternative splicing. (A) The two alternative 129-bp exons are adjacent in the mouse Ate1 gene. The thick line denotes genomic DNA; the striped and black rectangles denote the alternative 129-bp exons, M8 and M3 (see Materials and Methods); gray rectangles denote the flanking Ate1 exons, of unknown sizes; thin lines denote the alternative splicing patterns that yield the two forms of Ate1 mRNA. (B) The underlined 12 bp (6 bp in the intron and 6 bp in the exon) around the splice acceptor sites are identical between the two alternative 129-bp exons. (C) Scale diagrams of the two forms of mouse Ate1 cDNAs. The alternative 129-bp exons M8 and M3 are indicated by the striped and black boxes, respectively.

FIG. 5

Alternative splicing of Ate1 pre-mRNA in mammals (the mouse) but in neither plants (A. thaliana) nor arthropods (D. melanogaster). (A) The relevant Ate1 cDNA fragments from mouse (794 bp). A. thaliana (626 bp), and D. melanogaster (856 bp) were produced by RT-PCR (see Materials and Methods). The products were treated with the indicated restriction endonucleases that cut exclusively within the two alternative 129-bp exons of mouse Ate1 cDNAs (BspMI for Ate1-2; SexAI for Ate1-1) or within the corresponding regions of A. thaliana (PvuII and ScrFI) and D. melanogaster (AccI and EcoRV) Ate1 cDNAs. (B) The A. thaliana and D. melanogaster Ate1 genes. The exon-intron organization of these genes was deduced through comparisons of their cDNA sequences, determined in this work (see Materials and Methods), with the concurrently determined sequences of the corresponding genomic DNA regions (see text). The horizontal lines and rectangles denote, respectively, introns and exons, whose lengths are indicated below and above the line denoting introns. Thick horizontal lines indicate the regions of A. thaliana and D. melanogaster cDNAs that correspond to the alternative 129-bp exons of the mouse and human Ate1 cDNAs (Fig. 2 and 4). The lengths of the A. thaliana and D. melanogaster Ate1 genes are, respectively, ∼3 and ∼2.5 kb.

FIG. 6

The two forms of mouse ATE1p can implement the Asp/Glu-specific subset of the N-end rule pathway. (A) Relative enzymatic activities of βgal in ate1Δ S. cerevisiae transformed with plasmids expressing X-βgal (as Ub-X-βgal) test proteins (X = Met, Arg, Cys, Asp, or Glu) together with a plasmid expressing either yeast ATE1 (denoted as y), mouse ATE1-1p (denoted as 1), mouse ATE1-2p (denoted as 2), or the vector alone (denoted as v). The N-terminal residues of X-βgals in each set of experiments are indicated at the top. The activity of Met-βgal in cells transformed with vector alone is taken as 100%. (B) The two forms of mouse ATE1p exhibit no cooperativity in mediating the degradation of X-βgals. Shown are relative enzymatic activities of X-βgals in ate1Δ S. cerevisiae strain cotransformed with plasmids expressing X-βgals (Ub-X-βgals) (X = Met, Arg, Cys, Asp, or Glu) and the combinations of plasmids expressing the following proteins: mouse ATE1-1p and ATE1-1p (1+1), ATE1-1p and ATE1-2p (1+2), ATE1-2p and ATE1-2p (2+2), or two vector controls (v+v). One of the two vectors bore the TRP1 marker and the other bore the HIS3 marker (see Materials and Methods). Results are averages of four independent measurements, which differed by less than 10%.

FIG. 7

Intracellular localization of mouse ATE1-1p and ATE1-2p. Shown are green (GFP) fluorescence (a to d and f) and phase-contrast (e and g) micrographs of mouse NIH 3T3 cells transiently transfected with ATE1-1p-GFP (d to g) or ATE1-2p–GFP (a to c) fusion proteins (see Materials and Methods). Panels a to c show different examples of the exclusively cytosolic localization of ATE1-2p. Regions around the nucleus and in the lamellar protrusions at the edges of a cell (c) exhibit higher GFP fluorescence, possibly because of a greater thickness of cells in these areas. Panels d plus e and f plus g show pairs of GFP fluorescence and phase-contrast pictures of cells that express ATE1-1p-GFP. The cell in panels d and e shows ATE1-1p-GFP in the cytosol but not in the nucleus. Cells in panels f and g contain ATE1-1p-GFP in both the cytosol and the nucleus, the latter being apparently enriched in ATE1-lp-GFP.

FIG. 8

Northern hybridization analyses of mouse Ate1 mRNA. The Northern blots of mRNA from different mouse tissues were probed with an Ate1 cDNA fragment (nt 638 to 1734) which can hybridize to both forms of Ate1 mRNA. A mouse β-actin cDNA probe was used for comparing the total RNA loads as described previously (19). The same blot was also hybridized with mouse Ubr1 (29) (see the introduction). The apparent absence of Ate1 and Ubr1 mRNAs from the spleen is an artifact of RNA degradation in this lane of the blot (data not shown). Also shown are the results of analogous Northern hybridizations of the mouse Ntan1 cDNA, encoding the Asn-specific Nt-amidase (Fig. 1A), and E2_14K, encoding the relevant Ub-conjugating (E2) enzyme (19). The approximate sizes of transcripts are indicated on the right.