Affinity Isolation of Endogenous Saccharomyces Cerevisiae Nuclear Pore Complexes

Abstract

Studying protein complexes in vitro requires the production of a relatively pure sample that maintains the full complement, native organization, and function of that complex. This can be particularly challenging to achieve for large, multi-component, membrane embedded complexes using the traditional recombinant expression and reconstitution methodologies. However, using affinity capture from native cells, suitable whole endogenous protein complexes can be isolated. Here we present a protocol for the affinity isolation of baker's yeast (S. cerevisiae) nuclear pore complexes, which are ~50 MDa assemblies made up of 552 distinct proteins and embedded in a double-membraned nuclear envelope. Producing this sample allowed us for the first time to perform analyses to characterize the mass, stoichiometry, morphology, and connectivity of this complex and to obtain its integrative structure with ~9 Å precision. We believe this methodology can be applied to other challenging protein complexes to produce similar results.

Keywords: Affinity capture; Baker’s yeast; Electron microscopy; Endogenous macromolecular assembly; Native Isolation; Nuclear pore complex; S. cerevisiae; Structural and functional analyses.

Fig. 1. Affinity isolation methodology.

An outline of a general affinity isolation protocol, which can be optimized for various protein complexes from many organisms, as described in this work. (a) Cell lysis using flash freezing and cryo-milling to obtain cell powder which can be stored at −80°C and used as needed for any scale experiment. (b) Affinity capture by resuspending cell powder in an optimized affinity capture buffer and using the clarified (centrifuged and/or filtered) lysate for incubation with antibody-coupled magnetic beads to capture the complex on the beads followed by discarding the remaining supernatant and washing steps. Complex elution is accomplished by denaturation or by native elution (proteolytic removal or competitive binding) followed by separation from the beads. Eluted complex can be analyzed by SDS-PAGE and other downstream analyses mentioned in the text.

Fig. 2. SDS-PAGE analysis.

Typical results using the exact protocol described are shown (see section 3.5). Gel is loaded as described in section 3.5, 10 and scanned as described in section 3.5, 18. Lanes 1 and 5 contain a protein marker (molecular weights are indicated on the left side). Lanes 2 and 3 contain two Dynabeads pellet samples. Lane 4 contains the native elution sample. Lanes 6-10 contain BSA standards (25, 50, 100, 250, 500 ng, respectively). Black arrows indicate the “handle” protein MLP1 (see section 3.5, 19 and Note 14).

Fig. 3. Negative stain EM images.

Four representative images at different magnifications taken using grids prepared and imaged as described in section 3.6. Black boxes indicate a selection of individual NPCs. Magenta arrows point to central transporters within selected NPCs. Scale bar indicated in black in the bottom left side of every image.