Structural Organization of Human Full-Length PAR3 and the aPKC-PAR6 Complex.

ABSTRACT

The tripartite partition defect (PAR) polarity complex, which includes the proteins PAR3, atypical protein kinase C (aPKC), and PAR6, is a major regulator of cellular polarity. It is highly conserved and expressed in various tissues. Its largest component, PAR3, controls protein-protein interactions of the PAR complex with a variety of interaction partners, and PAR3 self-association is critical for the formation of filament-like structures. However, little is known about the structure of the PAR complex. Here, we purified non-filamentous PAR3 and the aPKC-PAR6 complex and characterized them by single-particle electron microscopy (EM). We expressed and purified an oligomerization-deficient form of PAR3, PAR3V13D,D70K, and the active aPKC-PAR6 dimer. For PAR3, engineering at two positions is sufficient to form stable single particles with a maximum dimension of 20 nm. aPKC-PAR6 forms a complex with a maximum dimension of 13.5 nm that contains single copies of aPKC. Thus, the data present a basis for further high-resolution studies of PAR proteins and PAR complex formation.