Date of Award
Fall 2023
Document Type
Dissertation
Degree Name
Doctor of Philosophy (PhD)
Department
Molecular, Cellular, and Developmental Biology
First Advisor
Bahmanyar, Shirin
Abstract
The endoplasmic reticulum (ER) is the largest membrane bound organelle in most cells. The ER is made up of distinct structural and functional domains, which include the nuclear envelope (NE) and peripheral ER sheets and tubules. The nuclear envelope surrounds and protects the genome, while the peripheral ER is essential for protein and de novo lipid synthesis. Lipids synthesized at the ER are either retained or shuttled to other membrane bound organelles through membrane contact sites or vesicular trafficking. As such, regulation of ER lipid synthesis is a tightly controlled process carried out by a subset of enzymes that are highly regulated. Defects in distinct steps of lipid synthesis have been implicated in obesity, insulin resistance and cancer. Here, I study lipid synthesis through CTDNEP1 (Sc Nem1) and its regulatory binding partner NEP1R1 (Sc Spo7) which control the activity of a key enzyme necessary for de novo glycerolipid synthesis, lipin 1 (Sc Pah1), in human cells. Regulation of lipin 1 by CTDNEP1 limits de novo glycerophospholipid synthesis throughout the cell cycle to restrict ER size and maintain NE shape. In my work, I show that deletion of CTDNEP1 impacts lipid synthesis due to the absence of a dephosphorylated pool of lipin 1. Mechanistically, I demonstrate that CTDNEP1 counteracts mTOR phosphorylation of lipin 1 in a cell cycle dependent manner. In addition, CTDNEP1 protects lipin 1 from proteasomal degradation. This work shows how CTDNEP1 regulates lipin 1 to limit ER lipid synthesis, yet it remained unknown how CTDNEP1 itself is regulated. Using a combination of structure-function analyses, in-silico studies, quantitative live and fixed cell microscopy, and biochemical studies, I show that NEP1R1 serves as a molecular membrane scaffold that limits proteasomal degradation of ER/NE-associated CTDNEP1. I find that CTDNEP1 contains an N-terminal predicted amphipathic helix that is necessary and sufficient for its localization to the ER/NE and to the surface of lipid droplets (LDs). NEP1R1 is a relatively stable protein, whereas ER/NE-associated CTDNEP1 is short-lived and targeted for degradation by the ubiquitin proteasome pathway. I then demonstrate that NEP1R1 temporarily stabilized CTDNEP1 by limiting its proteasomal degradation, which promotes CTDNEP1 activity to dephosphorylate lipin 1 and restrict ER size. Interestingly, I found a novel role for CTDNEP1 in restricting LD number independent of NEP1R1. These data reveal that distinct mechanisms control targeting to ER/NE membranes and protein stabilization of CTDNEP1, which is essential to the regulation of ER/NE lipid synthesis. Together this provides a framework into understanding how lipid synthesis is controlled through distinct protein regulatory mechanism to sustain proper lipid synthesis at the ER.
Recommended Citation
Carrasquillo Rodriguez, Jake Williams, "Mechanisms that Control Lipid Synthesis at the Endoplasmic Reticulum and Nuclear Envelope" (2023). Yale Graduate School of Arts and Sciences Dissertations. 1129.
https://elischolar.library.yale.edu/gsas_dissertations/1129
