The 3′ Overhangs at Tetrahymena thermophila Telomeres Are Packaged by Four Proteins, Pot1a, Tpt1, Pat1, and Pat2

Abstract

ABSTRACTAlthough studies with the ciliateTetrahymena thermophilahave played a central role in advancing our understanding of telomere biology and telomerase mechanisms and composition, the full complement ofTetrahymenatelomere proteins has not yet been identified. Previously, we demonstrated that inTetrahymena, the telomeric 3′ overhang is protected by a three-protein complex composed of Pot1a, Tpt1, and Pat1. Here we show that Tpt1 and Pat1 associate with a fourth protein, Pat2 (Pot1 associatedTetrahymena2). Mass spectrometry of proteins copurifying with Pat1 or Tpt1 identified peptides from Pat2, Pot1a, Tpt1, and Pat1. The lack of other proteins copurifying with Pat1 or Tpt1 implies that the overhang is protected by a four-protein Pot1a-Tpt1-Pat1-Pat2 complex. We verified that Pat2 localizes to telomeres, but we were unable to detect direct binding to telomeric DNA. Cells depleted of Pat2 continue to divide, but the telomeres exhibit gradual shortening. The lack of growth arrest indicates that, in contrast to Pot1a and Tpt1, Pat2 is not required for the sequestration of the telomere from the DNA repair machinery. Instead, Pat2 is needed to regulate telomere length, most likely by acting in conjunction with Pat1 to allow telomerase access to the telomere.