Detection of Nα-terminally formylated native proteins by a pan-N-formyl methionine-specific antibody

Abstract

N-formyl methionine (fMet)-containing proteins are produced in bacteria, eukaryotic organelles mitochondria and plastids, and even in cytosol. However, Nα-terminally formylated proteins have been poorly characterized because of the lack of appropriate tools to detect fMet independently of downstream proximal sequences. Using a fMet-Gly-Ser-Gly-Cys peptide as an antigen, we generated a pan-fMet-specific rabbit polyclonal antibody called anti-fMet. The raised anti-fMet recognized universally and sequence context-independently Nt-formylated proteins in bacterial, yeast, and human cells as determined by a peptide spot array, dot blotting, and immunoblotting. We anticipate that the anti-fMet antibody will be broadly used to enable an understanding of the poorly explored functions and mechanisms of Nt-formylated proteins in various organisms.

Keywords: N-formyl methionine; anti-fMet antibody; fMet/N-degron; mitochondrial methionyl-tRNA formyltransferase; peptide deformylase; protein synthesis.

Figure 1

Schematicrepresentation of the development of a pan-fMet-specific antibody.A, synthetic fMGSGC-peptide antigen for generation of a pan-fMet-specific (anti-fMet) antibody. The fMGSGC antigen contains Nt-formylated fMet, a flexible GSG linker, a thiol-based conjugatable Cys, and a KLH carrier protein. B, scheme for anti-fMet generation. Anti-fMet antibodies were induced by immunization of rabbits with the fMGSGC antigen, followed by antisera preparation, negative selection-based removal of nonspecific antisera, and subsequent protein A-affinity chromatography. fMet, N-formyl methionine; fMGSGC, fMet-Gly-Ser-Gly-Cys; GSG, Gly-Ser-Gly; KLH, keyhole limpet hemocyanin.

Figure 2

Broad binding specificity of anti-fMet for various Nt-formylated peptides.A, immunoblotting with the anti-fMet^Hs antibody purified through negative selection with HeLa cell lysates and SPOT peptide arrays with 11-residue peptides XZ-IAIGTYQEK (XZ-D2^3–11; X represents Met, fMet, or AcMet, Z is one of 20 amino acids, and D2^3–11 is derived from the 3–11 residues of C. reinhardtii D2 protein). B, dot immunoblotting with the anti-fMet^Hs antibody versus decremental amounts of the unmodified METSSENGSK peptide (11 Nt-residues of S. cerevisiae Pin4) and either its Nt-formylated or Nt-acetylated counterpart. C, same as (B), but with the MKVVKEFSGSK peptide (11 Nt-residues of S. cerevisiae Yjl068c). See also main text and Experimental procedures. A–C images are representative of two independent experiments. fMet, N-formyl methionine.

Figure 3

Broad binding specificity of anti-fMet for various Nt-formylated proteins.A, immunoblotting with anti-fMet^Hs and 0.2 μg MD-D2^3–11-GST purified from E. coli cells with or without 2 μg/ml actinonin treatment. B, same as (A), but with 0.2 μg Pgd1_HA-His6. C, immunoblotting with anti-fMet^Hs and 1 μM fMD-D2^3–11-GST that was preincubated with either 1 μM of EcPDF_His6 or catalytically inactive $EcPD{F}_{His6}^{E134A}$. D, same as (C), but with fPgd1_HA-His6 instead of fMD-D2^3–11-GST. E, immunoblotting with anti-fMet^Hs and purified unformylated MD-D2-GST (0.2 μg) or Nt-formylated fMD-D2-GST (0.2 μg) in the presence of 10 mM Met, fMet, or AcMet amino acids. The bottom immunoblot with anti-GST indicates equal amounts of loaded proteins. F, immunoblotting with anti-fMet^Hs and serially diluted MD-D2-GST or Nt-formylated fMD-D2-GST. G, same as (F), but with Pgd1_HA-His6 or Nt-formylated fPgd1_HA-His6. A–F images are representative of two independent experiments. EcPDF, Escherichia coli peptide deformylase; fMet, N-formyl methionine; Nt-, Nα-terminal.

Figure 4

Detection of Nt-formylated native proteins in E. coli, S. cerevisiae, and HeLa cells.A, immunoblotting with anti-fMet^Hs and extracts of ΔarcAB E. coli cells that were incubated in the either presence or absence of actinonin (2 μg/ml) for 0, 1, and 3 h. The bottom gel with Coomassie brilliant blue staining indicates equal amounts of loaded samples. B, immunoblotting with anti-fMet^Sc purified by negative selection with extracts of fmt1Δ S. cerevisiae cells that expressed $SCFmt{1}_{ha}^{\Delta SP}$ together with EcPDF or EcPDF^E134A. C, immunoblotting with anti-fMet^Hs and extracts (∼μg) of HeLa cells that expressed EcFMT_3f together with either EcPDF_3f or $EcPD{F}_{3f}^{E134A}$. A–C images are representative of two independent experiments. EcPDF, Escherichia coli peptide deformylase; fMet, N-formyl methionine; FMT, methionyl-tRNA formyltransferase; Nt-, Nα-terminal.

Figure 5

Detection of endogenous Nt-formylated native proteins in various human cell lines.A, immunoblotting with anti-fMet^Hs and extracts (15 μg) of HEK293 (embryonic kidney), HeLa (cervical carcinoma), MHCC97H (hepatocellular carcinoma), SW480 and HT29 (colorectal carcinoma), T24 and TCC (bladder carcinoma), and MDA231 and T47D (breast carcinoma) cells. A images are representative of three independent experiments. B, immunoblotting with anti-fMet^Hs and extracts (15 μg) of SW480 cells that were incubated in either the absence or presence of actinonin (10 μg/ml) for 3 h before cell lysis. Immunoblot with anti-tubulin was used as a loading control. C, subcellular fractions of SW480 cells, followed by immunoblotting with anti-fMet^Hs, anti-TOM20, and anti-tubulin. TOM20 and tubulin were used as cell compartment–specific markers for the mitochondrial and cytosolic fractions, respectively. B and C images are both representative of two independent experiments. D, identified fMet-containing protein from SW480 cells using Nt-peptide enrichment and subsequent LC-MS/MS. See also main text and Fig. S2. fMet, N-formyl methionine; Nt-, Nα-terminal.