Identification of a lumenal sequence specifying the assembly of Emp24p into p24 complexes in the yeast secretory pathway

Abstract

The p24 proteins are transmembrane proteins of the endomembrane system that play a poorly defined role in vesicle traffic between the endoplasmic reticulum and the Golgi apparatus. Various lines of evidence indicate that p24 proteins fall into four subfamilies (α, β, γ and δ) and that tetramers are assembled containing one representative from each subfamily; however, the nature of the protein-protein interactions within these hetero- oligomers is unknown. We have identified a lumenal segment of yeast p24β (Emp24p) that is necessary for its assembly into p24 complexes. Replacement of 52 C-terminal residues of Emp24p with the corresponding sequence from Erv25p (p24δ) generates a chimetic protein able to replace Emp24p in p24 complexes that retain partial function in vivo, ruling out a role for the transmembrane and cytosolic domains in specifying p24 interactions. Substitution of a further 50 residues, encompassing a heptad repeat region, abolishes the ability of the chimera to replace Emp24p but instead creates a protein that resembles its Erv25p parent in its requirement for stabilization by Emp24p. These data point to a role for coiled-coil interactions in directing subfamily-specific assembly of p24 oligomers that project into the lumen of transport vesicles, where they may act to exclude secretory cargo from coat protein complex type I-coated retrograde transport vesicles.