Enzyme-based reporters for mapping proteome and imaging proteins in living cells

Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Chemistry, 2013.Vita. Cataloged from PDF version of thesis.Includes bibliographical references.Each eukaryotic cell is exquisitely divided into organellar compartments whose functions are uniquely defined by the set of proteins they possess. For each individual protein, precise targeting to a specific sub-cellular location and trafficking between compartments are often key to its proper function. In fact, many human diseases are linked to mutations that cause mistargeting and/or defective trafficking. This thesis describes the development of enzyme-based reporters for measuring protein localization and trafficking. We employ two complementary approaches: a top-down approach, involving proteomics, to simultaneously acquire the subcellular localization information for hundreds of proteins; and a bottom-up approach, involving fluorescence imaging, to record detailed spatial information for proteins on an individual basis. This thesis is therefore divided into the following two parts. Part A describes a promiscuous protein labeling technique for proteomic mapping of organelles. This method capitalizes on peroxidase as a source of free radical generator. Compared to traditional sub-cellular fractionation methods, this novel approach obviates the need of organelle purification, thereby not only eliminating the potential artifacts associated with purification, but also greatly improving the temporal resolution of the proteomic mapping. Applying this technique to study the proteome of mitochondrial matrix and endoplasmic reticulum lumen has led to the discovery of several mitochondrial proteins whose localizations have previously been unknown or ambiguous. Part B discusses the development and application of site-specific protein labeling methods for studying receptor trafficking mechanisms. Building upon previous work in our lab, we utilized the Escherichia coli biotin ligase BirA and its acceptor peptide to site-specifically label the low-density lipoprotein receptor and studied its internalization and trafficking both at the ensemble imaging and single-molecule level. We discovered that this receptor internalizes as an oligomer into cells.by Peng Zou.Ph.D