Localization and orientation of the gamma-tubulin small complex components using protein tags as labels for single particle EM

Abstract

Gamma-Tubulin Small Complex (gamma-TuSC) is the universally-conserved complex in eukaryotes that contains the microtubule (MT) nucleating protein: gamma-tubulin. gamma-TuSC is a heterotetramer with two copies of gamma-tubulin and one copy each of Spc98p and Spc97p. Previously, the structure of gamma-TuSC was determined by single particle electron microscopy (EM) at 25A resolution. gamma-TuSC is Y-shaped with a single flexible arm that could be the key to regulating MT nucleation. EM gold labeling revealed the locations of gamma-tubulin at the top of the Y. In vivo Fluorescence Resonance Energy Transfer (FRET) suggested the relative orientations of Spc98p and Spc97p but did not distinguish which large subunit formed the flexible arm. Here, using fluorescent proteins as covalently attached tags, we used class averages and 3-D random conical tilt reconstructions to confirm the in vivo FRET results, clearly demonstrating that the Spc98p/97p C-termini interact directly with gamma-tubulin. Most significantly we have determined that the flexible arm belongs to Spc98p and our data also suggests that the N-termini of Spc98p and Spc97p are crossed. More generally, our results confirm that despite their small size, covalently-attached fluorescent proteins perform well as subunit labels in single particle EM.

Figure 1

A) Electron microscopy reconstruction of the S. cereviseae γ-TuSC. Light blue represents the flexibly attached arm and the more rigid body is presented in dark blue. The location of γ-tubulin was indicated by gold labeling, and the crystal structure of human γ-tubulin was manually fit into the structure (Kollman et al. 2008). B) A schematic representation of the arrengement of γ-TuSC proteins based on the relative orientations of N- and C- termini determined by in vivo FRET.

Figure 2

SDS-PAGE of γ-TuSC complexes. 1) γ-TuSC; 2) γ-TuSC:YFPSpc97p; 3) γ-TuSC:Spc97pYFP; 4) γ-TuSC:CFPSpc98p and 5) γ-TuSC:Spc98pCFP. The Tub4p His-tag linker is longer in the γ-TuSC, hence the Tub4p band migrates a bit slower than the other constructs. The band above Tub4p in the Spc98p-CFP constructs (Lanes 4,5) thought to be contamination, as it was observed on the EM samples. In addition, the ratios (see below SDS-PAGE) between proteins in each lane corresponded to the γ-TuSC stoichiometry: 2:1:1:1, Tub4p, Spc97p/98p tagged and untagged.

Figure 3

Structural analysis of tagged γ-TuSC complexes. A, B and C) Electron microscopy raw particles of γ-TuSC:Spc97pYFP, γ-TuSC:YFPSpc97p and γ-TuSC:YFPSpc98p respectively. The red arrow indicates the location of YFP and CFP density on each image. D, E and F) 2-D class averages of γ-TuSC:Spc97YFPp, γ-TuSC:YFPSpc97p and γ-TuSC:CFPSpc98p respectively with red arrows indicating the position of the tag. G and H) Overlay of the various 3-D reconstructions of γ-TuSC:Spc97YFPp and γ-TuSC:YFPSpc97p; the complex is shown in purple and the tags are colored red, blue, green and yellow. I) 3-D reconstruction of γ-TuSC:Spc97YFPp with the crystal structure of YFP, shown in yellow, manually docked in the protruding density. Size bar is 10nm.

Figure 4

A revised model of the S. cereviseae γ-TuSC based on observed tag positions. The flexibility of Spc98p is indicated with an arrow, and the the N-termini of Spc97p and Spc98p are crossed.