A study of monotopic-integrated proteins
Abstract/Contents
- Abstract
- The biological roles of integral membrane proteins are fundamentally related to their topologies. Bitopic and polytopic proteins have soluble domains on both sides of lipid membranes by adopting transmembrane domains (TMDs) and transfer information and solutes between aqueous compartments. Monotopic integral proteins (MIPs), in contrast, have all soluble domains facing one side of the membrane and organize membrane surfaces to facilitate biochemical reactions and biological processes. Although MIPs are critical to many cellular processes, detailed biochemical analyses required for their identification leave their structures under studied. One main factor that hinders systematic MIP study is that they are difficult to distinguish from TMD-containing proteins by primary sequence analysis and require biochemical assays on a protein-to-protein basis in a bilayer membrane. We exploited lipid droplets (LDs), which are monolayer-encased organelles that cannot accommodate bitopic or polytopic proteins, to unambiguously identify MIPs. Chapter 2 presents a method that uses chaotrope treatments, quantitative proteomics, and mixture modeling to identify integral membrane proteins in LD membranes, which would exclude bitopic and polytopic proteins, and thus, all be MIPs. We identified, 87 MIPs, which include proteins predicted to contain TMDs. We used a combination of biochemical solvent accessibility assays and coarse-grained molecular dynamics to identify a subset of the predicted TMDs in our dataset that adopt interfacial amphipathic helices. These results expand the set of definitive MIPs and suggest the importance of interfacial amphipathic helix motifs for anchoring MIPs to biological membranes. UBXD8 is a MIP that traffics from the endoplasmic reticulum (ER) to LDs. Although the trafficking mechanism from the ER to LD is unknown, the targeting of this protein to ER membranes was recently demonstrated to be critical for its localization to LDs. Farnesylated-Pex19 post-translationally delivers UBXD8 to Pex3, its ER membrane receptor. To determine which other LD proteins require farnesylated-Pex19 for LD localization, we combined chaotrope stripping with quantitative proteomics and identified four MIPs absent from LDs purified from cells expressing a farnesylation mutant but present in LDs purified from wild-type cells. This work expands our basic understanding of membrane biology by elucidating the identities and membrane topologies of MIPs. Furthermore, we identified novel MIPs dependent on Pex19 for trafficking to the ER membrane and subsequently to LDs. The methods presented herein can be applied to further detailed studies of MIPs and their biological functions.
Description
| Type of resource | text |
|---|---|
| Form | electronic; electronic resource; remote |
| Extent | 1 online resource. |
| Publication date | 2017 |
| Issuance | monographic |
| Language | English |
Creators/Contributors
| Associated with | Pataki, Camille Isabel | |
|---|---|---|
| Associated with | Stanford University, Department of Biochemistry. | |
| Primary advisor | Kopito, Ron Rieger | |
| Thesis advisor | Kopito, Ron Rieger | |
| Thesis advisor | Brandman, Onn | |
| Thesis advisor | Elias, Joshua | |
| Thesis advisor | Straight, Aaron, 1966- | |
| Advisor | Brandman, Onn | |
| Advisor | Elias, Joshua | |
| Advisor | Straight, Aaron, 1966- |
Subjects
| Genre | Theses |
|---|
Bibliographic information
| Statement of responsibility | Camille Isabel Pataki. |
|---|---|
| Note | Submitted to the Department of Biochemistry. |
| Thesis | Thesis (Ph.D.)--Stanford University, 2017. |
| Location | electronic resource |
Access conditions
- Copyright
- © 2017 by Camille Isabel Pataki
- License
- This work is licensed under a Creative Commons Attribution Non Commercial 3.0 Unported license (CC BY-NC).
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