Assembly and molecular architecture of PI4K IIa complexes

 

Miroslav Peterek1,2, Evžen Bouřa3, Daniel Němeček1,2

 

1Central European Institute of Technology, Masaryk University, Brno, CZ

2Department of Biochemistry, Max Planck Institute, Martinsried, DE

3Institute of Organic Chemistry and Biochemistry, Czech Academy of Sciences, Prague, CZ

 

The phosphatidylinositol 4-kinases (PI4K) catalyze the production of phosphatidyl inositol phosphates (PIPs) that regulate membrane associated signal transduction and protein trafficking in eukaryotic cells [1]. The PI4Ks are also hijacked by several RNA viruses to generate membranes enriched in phosphatidylinositide 4-phosphate lipids that are used as viral replication platforms [2]. Two types of PI4K's have been identified in mammalian cells: the type II kinases (α and β isoforms) that are stably associated with membranes and the type III kinases (α and β isoforms) that are soluble and remain in cytosol [3]. While type III kinases share homology to structurally well characterized PI3 kinases the structure of type II kinases is unknown.

We expressed and purified a recombinant PI4K IIa kinase and discovered that it can assemble into large multimeric complexes. Here, we present the initial structural analysis of these complexes that were imaged by negative stain and cryo electron microscopy. Electron micrographs showed heterogeneous particles of globular shape ranging from 110 to 310 Å in diameter. The particles were separated into three groups based on their overall diameter, aligned and classified in 2D using EMAN package. Most particles (~70%) appeared as small compact spheres of ~110 Å diameter. About 17% particles had an oval shape with dimensions ~250 x 200 Å. Interestingly, about 13% of the particles formed large rings with ~310 Å outer diameter, ~140 Å thickness and ~90 Å inner hole. While the small particles likely represent a tight assembly of several PI4KIIα molecules (54 kDa), the two larger particles exhibit similar architecture as some other membrane binding proteins – respectively, the intermediate particles appear as compact assemblies of the human caveolin-3 protein [4] and the large rings are not dissimilar to assembled ESCRT proteins at the necks of budding viruses from the cell membrane [5].  

 

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