The overall goal of my project is to understand how the molecular chaperone Lhs1 facilitates the degradation of the Epithelial Sodium Channel (ENaC), an ion channel on the cell membrane that plays an important role in sodium reabsorption in the kidney and lungs. Specifically, I am studying how mutations in different domains of Lhs1 affect how Lhs1 facilitates ENaC degradation. Degradation, and ER-associated degradation (ERAD) in particular, is critical in removing misfolded proteins and regulating protein levels in the cell.
One aspect of my project that I have been working on this semester pertains to a double deletion strain, ∆sil1∆lhs1. In yeast, Sil1 and Lhs1 are both ER lumenal nucleotide exchange factors (NEFs) for the ER lumenal Hsp70, Kar2. In brief, Kar2 plays a role in a variety of protein folding processes and requires NEFs, such as Sil1/Lhs1, to release bound ERAD substrates and allow them to fold properly or be degraded. Although Sil1 and Lhs1 are functionally redundant, previous work indicates that the ∆sil1∆lhs1 double deletion is synthetic lethal. In other words, the Sil1 and Lhs1 deletions on their own do not result in cell death but the two deletions together do. One of the ways we can study the double deletion in yeast is by transforming Lhs1 on a methionine repressible promoterinto ∆sil1∆lhs1 cells. Therefore, when we add methionine to ∆sil1∆lhs1 cells expressing Lhs1, we repress Lhs1 expression and can now examine the effects of the ∆sil1∆lhs1 double deletion. Additionally, it is necessary to use a truncated version of Lhs1 (which has its endoplasmic reticulum (ER) retrieval sequence removed) to destabilize the protein and sensitize it to the methionine treatment. I first confirmed that the ∆sil1∆lhs1 double mutation is synthetic lethal, by growing ∆sil1∆lhs1 with full length and truncated Lhs1 on medium lacking methionine (-His+glu) and medium with methionine (YPD + Met). As shown in Figure 1 below, ∆sil1∆lhs1 + truncated Lhs1 cells exhibit a synthetically lethal defect on medium containing methionine. Previous research also indicates that ∆sil1∆lhs1 cells exhibit a severe translocation defect; the double deletion impedes the cells’ ability to move proteins across the ER membrane, indicating that Sil1 and Lhs1 play a role in the translocation process. I next confirmed that we could visualize the translocation defect. Therefore, I treated the ∆sil1∆lhs1 + full length/truncated Lhs1 strains with methionine, prepared whole cell extracts for SDS-PAGE and Western blotting, and immunoblotted for Kar2 and PDI. As evidenced in Figure 2 below, ∆sil1∆lhs1 + truncated LhsI cells accumulate the secretory precursors prePDI and preKar2 and thus exhibit a translocation defect 3 hours after the methionine treatment.
The next step in regard to this aspect of my project is to successfully transform Lhs1 mutants into ∆sil1∆lhs1 cells, which will allow me to determine if the mutants rescue growth and the translocation defect and to what extent, in comparison with wild-type Lhs1. I have already cloned the Lhs1 mutants, which include Lhs1 ∆Loop, Lhs1 ∆CT, and Lhs1 D26A, onto a Ura marked plasmid. After I transform the Lhs1 mutants on the Ura plasmid into ∆sil1∆lhs1 + truncated Lhs1 cells, I can treat the cells with methionine to repress expression of truncated Lhs1. As a result, I will be left with ∆sil1∆lhs1 cells that are expressing an Lhs1 mutant. However, this transformation has been particularly challenging. The first attempt at the transformation was unsuccessful, as there was no growth on any of the plates. After talking with my research mentor, we adjusted the transformation protocol to account for the additional stress that the double deletion induces in cells and reattempted the transformation; nonetheless, the transformation was unsuccessful again.
This challenging transformation is one example of how my understanding of the research process has changed as I have been conducting research this semester. I have learned that the research process is not a simple stepwise process, as I once learned in my high school science classes. Rather, more times than not, an experiment or protocol does not work on the first attempt. Instead, you must make slight adjustments to the experiment until it is successful. Therefore, I have come to understand that patience is extremely important in the research process, and I am grateful to have wonderful research mentors, who provide me with guidance and support as I work through challenges that arise. In addition, I have gained a greater appreciation for the collaborative nature of the research process and the research field in general. Working in a space surrounded by researchers who have an immense knowledge of the field means that there is no problem that I (or anyone else, for that matter) am forced to solve alone. Everyone is always willing to help each other out and work through challenges together, and this is one aspect of conducting research that I particularly enjoy.
Now that the CURF is over, I am going to continue to work on my project, in preparation for completing an Honors Thesis in the Department of Biological Sciences. My work will continue to expand upon the research that I have done this semester, and I look forward to seeing where my project will take me!
Figure 1. ∆sil1∆lhs1 cells expressing truncated Lhs1 are not viable on methionine-containing medium.
Figure 2. Expression of truncated Lhs1 in ∆sil1∆lhs1 cells results in translocation defect after methionine treatment.
