Production and initial characterization of Dad1p, a component of the Dam1-DASH kinetochore complex.
In all dividing eukaryotic cells, the mitotic spindle (composed primarily of microtubules) must interact with chromosomes through a complex protein assembly called the kinetochore. In Saccharomyces cerevisiae, the Dam1-DASH complex plays an important role in promoting attachment between the kinetochore and the mitotic spindle. It also actively participates in the physical separation of sister chromatids in anaphase. Understanding the biochemical mechanisms used by Dam1-DASH has been facilitated by bacterial co-expression of the ten Dam1-DASH genes, which results in the production of a heterodecameric protein complex that can be studied in vitro. However, individual protein subunits are not soluble when expressed in E. coli, thus precluding analysis of the nature of the interaction between subunits and an examination of the assembly of the functional complex. In this paper, we describe the expression, solubilization, purification and refolding of Dad1p, one of the Dam1-DASH complex subunits. In addition, we show that Dad1p, when isolated in this manner forms dimers and/or tetramers, dependent upon protein concentration. This work provides an important tool for studying the Dam1-DASH complex that was previously unavailable, and provides an avenue of investigation for understanding how the individual heterodecamers associate with each other to facilitate chromosome segregation.
Waldo
Jennifer
J
Department of Biology, SUNY New Paltz, New Paltz, New York, United States of America. waldoj@newpaltz.edu
Scherrer
Michael
M
D018797:Cell Cycle Proteins / Q000378:metabolism*; D004926:Escherichia coli; D006863:Hydrogen-Ion Concentration; D018386:Kinetochores / Q000378:metabolism*; D008869:Microtubule-Associated Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D046912:Multiprotein Complexes / Q000378:metabolism*; D017510:Protein Folding; D012441:Saccharomyces cerevisiae / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D012995:Solubility
D018797:Cell Cycle Proteins; C116003:DAM1 protein, S cerevisiae; C439311:Dad1 protein, S cerevisiae; D008869:Microtubule-Associated Proteins; D046912:Multiprotein Complexes; D029701:Saccharomyces cerevisiae Proteins