Identification of Specific Genes Involved in Proteasome Autophagy

Undergraduate #57
Discipline: Biological Sciences
Subcategory: Cell and Molecular Biology

Ruben Pando - Seward County Community College
Co-Author(s): Kenrick Waite, Gabrielle Vontz, and Jeroen Roelofs, Kansas State University, Manhattan, KS

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The proteasome is a complex protease containing 66 subunits. In eukaryotic cells, it is responsible for the degradation of shortlived and damaged proteins, thereby ensuring overall health and viability of cells. The proteasome is comprised of two subcomplexes; the regulatory particles that recognize ubiquitinated proteins and the core particle where degradation occurs. While the proteasome is a very stable complex, cells need to be able to degrade it when it is damaged or too abundant in the cell. We recently identified a process that targets proteasomes to the yeast vacuole for degradation. In this study, we hypothesize that specific genes such as Rpn10 and Mak10 are involved in this process, called proteasome autophagy or proteaphagy. To test this, we deleted the genes from yeast by replacing them with a selection cassette. An initial polymerase chain reaction allowed us to create a DNA fragment containing the hygromycin selection cassette flanked by fragments of DNA that will target the cassette to the gene of interest. Upon purification of the DNA fragment, we used it to transform yeast strains that already contained a GFP-tagged proteasome subunit. The tagged proteasome subunit is required for the assay that determines vacuolar targeting of the proteasomes. The hygromycin resistant transformants we obtained were analyzed for proper integration of the DNA fragment. For this, we first purified the genomic DNA, followed by PCR analyses to confirm the absence of the specific gene and the proper integration of the selection cassette. Next, strains were analyzed for their ability to perform proteasome autophagy. A nitrogen starvation experiment demonstrated that proteasome autophagy still occurred without Rpn10 or Mak 10 present. This result yields further understanding of how the proteasome functions and degrades. The analyses of these and other genes will provide us with the crucial information of which genes are required for targeting proteasomes to the vacuole for degradation and thus regulate the levels of this important cellular machine.

Funder Acknowledgement(s): This material is based upon research supported by the National Science Foundation (grant 1305059) in accordance with the Kansas Louis Stokes Alliance for Minority Participation. Additional research made possible by the National Institutes of Health NIGMS (grant 1R15GM112142).

Faculty Advisor: Jeroen Roelofs,