High-throughput investigation of protein localization and protein-protein interaction with a light-gated transcriptional reporter
Abstract/Contents
- Abstract
- High-throughput screens of biological behaviors and functions have been revolutionized by the growth of computer and data sciences. By leveraging the power of computers to rapidly measure and record an experimental value and statistically analyze the results across thousands or millions of measurements, biologists are able find novel functions and identify subtle trends at genome-scale. In the field of genetics, CRISPR-based gene disruption and next-generation sequencing provide for the rapid, robust identification of genes which influence a cellular function. In the field of proteomics, massive datasets have been produced to identify and characterize the interactions of proteins. However, there are limitations to the power of high-throughput screens. The key bottleneck of most screens is the juncture at which biology is translated into code. The boundaries to the science we can explore in high-throughput are formed by our ability to convert biology of interest into an easily-measured and easily-analyzed readout. Here, I describe protein-engineering efforts to expand range of biological questions that can be studied in high-throughput screens with next-generation sequencing analysis. In the first chapter, I describe the modification of a light- and interaction-gated transcriptional reporter to detect of protein localization with transcriptional readout. Pooled gene perturbation screens, in which all cells are in a shared culture, are limited to simple readouts such as cell proliferation or fluorescence-activated cell sorting (FACS) to isolate large numbers of phenotypic hits. There exist few approaches to screen for complex biological functions which are generalizable to more than one cellular process. We developed HiLITR as a genetically encoded reporter which converts colocalization of two protein components into proteolytic release of a membrane-bound transcription factor. We combined HiLITR with CRISPRi screening to identify factors which influence the trafficking of mitochondrial and ER tail-anchored proteins. We find that the loss of the SUMO E1 ligase SAE1 results in mislocalization and destabilization of mitochondrial tail-anchored proteins. We further demonstrate that EMC10 of ER membrane complex has a distinct role opposing the transmembrane-domain insertase activity of the complex. By transcriptional readout of complex processes, HiLITR expands the scope of functional genomics screening technologies to broad questions of protein localization and trafficking. In the second chapter, I describe the adaptation of the light- and interaction-gated transcriptional reporter for the high-throughput identification of novel protein-protein interactions (PPI) with temporal resolution. Currently, high-throughput approaches to detect time-variant PPI are limited to readout with mass spectrometry. To enable such screens with sequencing readout, we created the SPARK two-hybrid platform, in which a bait protein fused to a light-gated transcription factor is probed with a library of protease-tagged proteins. Interaction between bait and prey releases the transcription factor, producing fluorescence protein expression which can be processed by FACS. A genetic record of the interacting prey proteins can then be recovered from collected cells. We performed a SPARK two-hybrid screen on the beta-2 adrenergic receptor before, during, and after activation with agonist, identifying activation- and time-dependent interactions with the receptor, including both well-studied and potentially novel interactions. By providing for analysis via sequencing, SPARK two-hybrid offers a new means of time-resolved PPI-detection that is complementary to existing, proteomics-based approaches.
Description
| Type of resource | text |
|---|---|
| Form | electronic resource; remote; computer; online resource |
| Extent | 1 online resource. |
| Place | California |
| Place | [Stanford, California] |
| Publisher | [Stanford University] |
| Copyright date | 2021; ©2021 |
| Publication date | 2021; 2021 |
| Issuance | monographic |
| Language | English |
Creators/Contributors
| Author | Coukos, Robert |
|---|---|
| Degree supervisor | Ting, Alice Y |
| Thesis advisor | Ting, Alice Y |
| Thesis advisor | Bassik, Michael |
| Thesis advisor | Fordyce, Polly |
| Thesis advisor | Kobilka, Brian K |
| Degree committee member | Bassik, Michael |
| Degree committee member | Fordyce, Polly |
| Degree committee member | Kobilka, Brian K |
| Associated with | Stanford University, Department of Genetics |
Subjects
| Genre | Theses |
|---|---|
| Genre | Text |
Bibliographic information
| Statement of responsibility | Robert Coukos. |
|---|---|
| Note | Submitted to the Department of Genetics. |
| Thesis | Thesis Ph.D. Stanford University 2021. |
| Location | https://purl.stanford.edu/jm247mt1272 |
Access conditions
- Copyright
- © 2021 by Robert Coukos
- License
- This work is licensed under a Creative Commons Attribution Non Commercial 3.0 Unported license (CC BY-NC).
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