Oligomerization of peptides analogous to the cytoplasmic domains of coatomer receptors revealed by mass spectrometry.
Members of the p24 family of type I transmembrane proteins are involved in budding of coat protein type I (COPI)-coated vesicles. They serve as coat protein receptors, binding via their cytoplasmic domains to coatomer, a stable cytosolic protein complex that represents the major coat component of these vesicles. Experimental evidence suggest that p23, a member of the p24 family, binds to coatomer in an oligomeric state and that this binding triggers polymerization of the coat protein. Toward an understanding of this process at the molecular level, formation of noncovalent complexes and their relative stabilities were analyzed by Fourier transform ion cyclotron resonance mass spectrometry using nanoelectrospray ionization. Specificity and stability of oligomers formed were established to depend on characteristic peptide sequence motifs and were confirmed by mass spectrometric competition experiments with control peptides. Mutations in the peptide sequence caused decreased interaction and destabilization of the noncovalent complexes. The formation and relative stabilities of dimeric and tetrameric complexes were assessed to be formed by cytoplasmic tails of coatomer receptors. The direct molecular identification provided by mass spectrometry correlates well with biochemical results. Thus, electrospray ionization mass spectrometry proves to be a powerful tool to investigate physiologically relevant peptide complexes.
Fligge
T A
TA
Fachbereich Chemie, Universität Konstanz, Germany.
Reinhard
C
C
Harter
C
C
Wieland
F T
FT
Przybylski
M
M
D000595:Amino Acid Sequence; D020755:Coat Protein Complex I / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D020756:Coatomer Protein / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D004355:Drug Stability; D013058:Mass Spectrometry / Q000379:methods; D009842:Oligopeptides / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D020836:Protein Structure, Quaternary; D017434:Protein Structure, Tertiary
D020755:Coat Protein Complex I; D020756:Coatomer Protein; D009842:Oligopeptides