Figure 7.

Scm3^Sp Associates with Itself, CENP-A^Cnp1, and Mis18

(A) Coaffinity purification of Scm3^Sp and CENP-A^Cnp1. Strains expressing the following tagged protein(s) were analyzed: Scm3-13myc only, both Scm3-13myc and CENP-A^Cnp1-CTAP, CENP-A^Cnp1-GFP only, or both CENP-A^Cnp1-GFP and Scm3-CTAP. Tagged genes were expressed with the native promoters at their endogenous loci, except that CENP-A^Cnp1-GFP was overexpressed (labeled with “oe”). IgG beads were used for affinity purification from cell extracts. Input samples (I) and bead-bound samples (B) were subject to western blotting using the indicated antibodies.

(B) In vitro binding assay. (Left) ³⁵S-labeled Scm3^Sp was produced by in vitro transcription-translation in reticulocyte lysate (see [C], right panel) and incubated with GST, GST-Cnp1, or GST-H3. Complexes were pulled down with glutathione agarose, washed, and analyzed by SDS-PAGE and fluorography.

(C) ³⁵S-labeled Sim3, Scm3^Sp, Mis16, and Mis18 were produced (right panel) and used in vitro binding assay with GST or GST- Scm3^Sp (left panel).

(D) Model for Scm3^Sp function. The Mis6/Sim4 complex and Mis16/Mis18 are required to recruit Scm3^Sp to centromeres. Scm3^Sp acts as a receptor at the centromere for incoming CENP-A^Cnp1 from the Sim3 escort-chaperone. In conjunction with Mis16/Mis18 and other factors, Scm3^Sp mediates the incorporation of CENP-A^Cnp1 (C) in subkinetochore chromatin in place of H3.