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 p rotein receptors, binding via their cytoplasmic domains to coatomer, a stab le cytosolic protein complex that represents the major coat component of th ese vesicles. Experimental evidence suggest that p23, a member of the p24 f amily, binds to coatomer in an oligomeric state and that this binding trigg ers polymerization of the coat protein. Toward an understanding of this pro cess at the molecular level, formation of noncovalent complexes and their r elative stabilities were analyzed by Fourier transform ion cyclotron resona nce mass spectrometry using nanoelectrospray ionization. Specificity and st ability of oligomers formed were established to depend on characteristic pe ptide 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. Th e formation and relative stabilities of dimeric and tetrameric complexes we re assessed to be formed by cytoplasmic tails of coatomer receptors. The di rect 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 peptid e complexes.