Live cell proximity biotinylation enables proteomic analysis of mitochondria-endoplasmic reticulum contact sites

• Main Author: Hung, Victoria
• Other Authors: Alice Y. Ting.
• Format: Others
• Language: English
• Published: Massachusetts Institute of Technology 2016
• Subjects: Chemistry.
• Online Access: http://hdl.handle.net/1721.1/105026

Thesis: Ph. D., Massachusetts Institute of Technology, Department of Chemistry, 2016. === Cataloged from PDF version of thesis. === Includes bibliographical references. === The cell is able to carry out an astounding number of biological functions in part due to its ability to compartmentalize chemical reactions to different cellular regions and organelles. In order to understand the biological pathways that occur in a specific cellular region, one needs to know the identity of the protein players and how they behave spatially and temporally in the context of a living cell. Obtaining complete protein inventories is a recurring challenge in cell biology, and although mass spectrometry-based proteomics has made great strides in cataloging all proteins, traditional protocols are limited because they rely on a purification of the cellular entity of interest. This purification step is imperfect, and furthermore, many cellular regions cannot be purified. Prior work in the Ting lab developed a methodology to bypass organelle and complex purification by using an engineered ascorbate peroxidase (APEX) to tag proximal endogenous proteins with biotin for subsequent purification and identification by mass spectrometry. Although this method worked well in membrane-enclosed compartments, it was insufficiently specific when applied to unbounded or partially bounded regions such as the mitochondrial intermembrane space. This thesis presents a ratiometric tagging strategy that can achieve high spatial specificity in unbounded cellular regions. We use this strategy to map the proteomes of mitochondrial subcompartments to understand how the mitochondrion communicates with the rest of the cell, and in particular, with the endoplasmic reticulum. These proteomes led to the identification of a protein that may be a component of a tethering complex between mitochondria and the endoplasmic reticulum. === by Victoria Hung. === Ph. D.