Proteomic investigation of protein interactions and post-translational modifications of the Pfh1 helicase and yeast telomerase holoenzymes

Abstract

In this thesis I describe a targeted proteomics approach to study in vivo protein interactions and post-translational modifications of protein complexes involved in the maintenance of chromosome stability. Specifically I have used mass spectrometry to study the Schizosaccharomyces pombe PIF1 family helicase, Pfh1 and the telomerase enzyme of S. pombe and Saccharomyces cerevisiae.

Similarly to higher eukaryotes, S. pombe encodes a single PIF1 family protein, Pfh1. Nuclear and mitochondrial isoforms of Pfh1 play essential roles in the maintenance of chromosomal and mitochondrial DNA, respectively. Immunoaffinity purification (IP) of Pfh1-GFP and quantitative mass spectrometry establish a functional role of Pfh1 in DNA replication and telomere regulation. Mass spectrometry revealed S-phase enrichment of Pfh1 protein interactions that have a critical role in DNA replication including the DNA leading strand replication polymerase (Pol2), and telomere regulation including the RecQ family helicase Rqh1. Furthermore, cells overexpressing Pfh1 display longer telomeres compared to cells expressing only the mitochondrial isoform of Pfh1. These findings support a model that Pfh1 facilitates replication fork progression at the telomeres and positively regulates telomere length.

Second, I have purified telomerase from S. pombe and S. cerevisiae with the goal of identifying novel protein components and post-translational modifications. The telomerase holoenzyme is composed of a templating RNA, a catalytic reverse transcriptase protein subunit, and associated proteins important for its biogenesis, activity, and recruitment to the telomere. Immunopurificaiton of telomerase was accomplished by overexpression of multiple protein components with a GFP epitope tag. In both organisms, overexpression of telomerase resulted in a lengthening of telomeres, suggesting a functional overexpressed complex. In S. pombe, I have confirmed several protein associations known to play a functional role in telomere maintenance as well as identified a novel interaction with an essential component of the mRNA nuclear export pathway, Ptr1. A ptr1-1 mutation revealed shorter telomeres, suggesting a functional role in telomere maintenance. Data supports a model that Ptr1 functions in telomerase RNA nuclear export and assembly with telomerase protein components. S. cerevisiae proteomic experiments are in progress and have thus far identified a novel phosphorylation site of the telomerase holoenzyme protein Est1 at S644.

Supplemental Tables 1-5: MS protein interaction data