Steady-State Regulation of Secretory Cargo Export and ER Homeostasis by Inositol Trisphosphate Receptors and Penta-EF-Hand Proteins

Author

Aaron J. Held, University of Montana, MissoulaFollow

Year of Award

2023

Document Type

Dissertation

Degree Type

Doctor of Philosophy (PhD)

Degree Name

Cellular, Molecular and Microbial Biology

Department or School/College

Division of Biological Sciences

Committee Chair

Jesse Hay

Commitee Members

Mark Grimes, Ekaterina Voronina, Richard Bridges, Scott Wetzel, John Quindry

Keywords

IP3 Receptors, PEF proteins, ER-to-Golgi, Calcium, MAMs, ER Homeostasis

Subject Categories

Biological Phenomena, Cell Phenomena, and Immunity | Medical Cell Biology | Medical Molecular Biology

Abstract

Constant protein degradation and turnover necessitates constitutive secretion that delivers the correct mix of nascent proteins to their appropriate subcellular destinations. Cells thus exhibit steady-state secretion and the additional ability to adjust secretory flux, though we lack a clear understanding of this critical process. During secretion, the COPII coat is responsible for providing a balance of actively and passively selected ER cargos to enter the secretory pathway. Furthermore, Ca²⁺ -binding proteins have been implicated in regulating this process in response to Ca²⁺ signals. In Chapter 1, we review the secretory pathway and vesicular trafficking, with a focus on ER-to-Golgi transport. We then examine the known roles of Ca²⁺ in ER proteostasis and trafficking with a particular emphasis on the Inositol 1,4,5-Trisphosphate Receptors (IP3Rs) and the Penta-EF-Hand (PEF) proteins ALG-2 and Peflin. Chapter 2 describes experimental procedures while Chapter 3 examines the roles of Ca²⁺ and the PEF proteins in determining steady-state trafficking flux. We find that in Normal Rat Kidney (NRK) epithelial cells, depletion of the IP3R-3 isoform augments Ca²⁺ signaling, including spontaneous Ca²⁺ oscillations. This shift in Ca²⁺ signaling drove an ‘activated’ ALG-2 phenotype in which ER-to-Golgi transport of the model cargo VSV-G is accelerated. Additionally, we find that ALG-2 activation augments COPII cargo sorting stringency, concentrating COPII client cargo at ER Exit Sites (ERES) at the expense of bulk flow cargos. These findings suggest that the steady-state transport and cargo sorting capacity of a given cell may be influenced by its spontaneous Ca²⁺ signaling characteristics via ALG-2 regulation of COPII function. Chapter 4 details a set of preliminary experiments exploring the role that IP3R-3 and Mitochondria-ER Contact Sites (MERCs or MAMs) may play in the maintenance of ER proteostasis. Depletion of IP3R-1 from NRK cells–a condition which favors function of the IP3R-3 channel–causes partial depletion of ER Ca²⁺, yet cells are protected from ER stress. This is of interest since Ca²⁺ is traditionally thought of as a rate-limiting component for protein folding in the ER. Since IP3R-3 is known to be the IP3R at MAMs, this data highlights the protective effects of MAMs on ER proteostasis.

Recommended Citation

Held, Aaron J., "Steady-State Regulation of Secretory Cargo Export and ER Homeostasis by Inositol Trisphosphate Receptors and Penta-EF-Hand Proteins" (2023). Graduate Student Theses, Dissertations, & Professional Papers. 12188.
https://scholarworks.umt.edu/etd/12188