Journal
MOLECULAR BIOTECHNOLOGY
Volume 64, Issue 12, Pages 1319-1327Publisher
SPRINGERNATURE
DOI: 10.1007/s12033-022-00504-1
Keywords
PAR3; PAR6; aPKC; PAR complex; Polarity; Single-particle electron microscopy
Funding
- Danish Council for Independent Research
- Fabrikant Einar Willumsens Mindelegat
- Lundbeck Foundation's Fellowship program
- Sapere Aude Program of the Danish Council for Independent Research
- Henny og Sophie Clausen og mobelarkitekt Aksel Clausens Fond
- Villum Foundation
- Projekt DEAL
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The tripartite partition defect (PAR) polarity complex is a major regulator of cellular polarity, consisting of PAR3, atypical protein kinase C (aPKC), and PAR6 proteins. This study characterized PAR3 and the aPKC-PAR6 complex using single-particle electron microscopy (EM), providing a basis for further high-resolution studies of PAR proteins and complex formation.
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, PAR3(V13D,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.
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