(A) Top row: individual images of 10% 2D gels (pH range 3.5–10 increases left to right) of whole cell extracts from wild-type (+/+, WT) cells labeled with Cy2 (1, left), WT cells treated with proteasome inhibitor labeled with Cy3 (2, middle), and arginylation-free Ate1 ^−/− cells labeled with Cy5 (3, right). Bottom row: pairwise comparison of the gels shown on top in different fluorescence channels. Colors were altered to red and green to allow better visual comparison.
(B) Quantification of the number of spots up- or down-regulated in response to each treatment. See also . Proteins identified in the most abundant spots from Coomassie-stained gels obtained under similar conditions are listed in (reflecting the comparison between gels 1 and 3) and S2 (reflecting the comparison between gels 1 and 2). See explanations in the text.

Autoradiographs of wild-type (+/+) and Ate1 ^−/− (−/−) whole cell extracts treated with cycloheximide in medium containing ¹⁴C-Arg, fractionated on 2D gels. Spots circled in red in the wild-type gel show increase in Arg incorporation above that in the Ate1 ^−/− cells, suggesting that Arg in these spots is incorporated posttranslationally. Proteins identified in the most abundant of the up-regulated spots are listed in .

Top: antibody generation strategy showing the sequences of the peptides used for antibody production and immunodepletion to ensure antibody specificity for Arg in the N-terminal position. Bottom: dot blot of the affinity-purified antibodies to N-terminal Arg–Asp and Arg–Glu sequences against the peptides listed on the top.

Peptide sequences are shown on top of each spectrum, with the added Arg boxed in red. Observed b and y ions are shown in red and blue, respectively on the MS/MS spectra and in the corresponding tables for each spectra. Generic names of the proteins are shown underneath the corresponding spectra. The spectra show peptides arginylated on N-terminal alanine (Ala), Leu, Gly, and phenylalanine (Phe) residues, all of which were previously believed to be unable to serve as an Arg acceptor site. Precursor mass accuracies for the shown spectra are 2.1 ppm (Ala), 3.8 ppm (Leu), 8.0 ppm (Gly), and 5.6 ppm (Phe). See for accession numbers and arginylated positions and for larger scale images of the MS/MS spectra for all identified residues.

Crystal structure (left) and primary amino acid sequence (right) of 8-kDa dynein light chain with the arginylated position marked in yellow on the polypeptide chain and denoted with red letter R. Arginylation results in modification of the protein surface, expected to affect molecular interactions and possibly association with the dynein polypeptide complex.

Three-dimensional structures of proteins downloaded from the National Center for Biotechnology Information structure database at http://www.ncbi.nlm.nih.gov/ (PDB identifiers 1HLU for beta actin, 1TUB for tubulin, 1DXT for hemoglobin, and 1ZNQ for GADPH) with arginylated positions highlighted in yellow within the polypeptide chains and marked with red letters R. Alpha and beta for tubulin indicate the corresponding polypeptide chains. See Text S1 for explanation of the functional significance of the arginylated sites in each shown protein.

Top: schematic representation of a protein containing two subunits (I, magenta and brown, and II, blue), whose subunit I is divided into two structurally and functionally distinct subdomains (A and B) within the same polypeptide chain. Middle: proteolytic products of this protein produced by degradation (left), interdomain cleavage (middle), or hypothetical intrachain proteolysis that occurs without the loss of the tertiary structure (right). Bottom: arginylation of the N-termini exposed during these proteolytic events could either produce denatured arginylated peptides (left), modulate the surface properties of active protein fragments (middle), or modulate the surface properties of the intact molecule (right). The last case also includes side chain arginylation, where Arg could be added onto the side chain of Asp or Glu (or possibly other residues) without proteolytic cleavage. Letters R in different colors indicate Arg residues added onto the damaged protein products (white), functional sites on the surface of proteins or domains (red), or side chains exposed on the protein surface (green).