Year of Award

2021

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, Brent Ryckman, J. Stephen Lodmell, Michael Kavanaugh

Keywords

Apoptosis-linked gene 2 (ALG-2), Calcium, Calcium signalling, COPII, Peflin, Vesicle trafficking

Abstract

The constitutive secretory pathway influences almost every aspect of cellular function: generation and maintenance of subcellular compartments; cell growth and differentiation; and production and deposition of the extracellular matrix. Classically, constitutive trafficking was thought to be continuous and unregulated—in contrast to regulated secretion, wherein vesicles are stored intracellularly until a Ca2+ signal causes synchronous membrane fusion. However, early studies demonstrated a Ca2+ requirement for endosome fusion, ER to Golgi, and intra- Golgi trafficking, demonstrating that Ca2+ is a regulator of constitutive trafficking steps as well. In Chapter 1, I review recent evidence for Ca2+ involvement and the specific Ca2+-related machinery implicated in trafficking events throughout the constitutive trafficking systems. Notably, I describe a series of Ca2+ pumps, channels, and Ca2+-binding effector proteins—and their trafficking machinery targets—that together regulate the flux of cargo in response to genetic alterations or baseline and agonist-dependent Ca2+ signals. Of the intracellular constitutive trafficking steps, the ER-to-Golgi transport stage is the busiest, transporting up to one-third of all eukaryotic proteins. However, the potential regulation of these steps by Ca2+ signaling events has remained largely uncharacterized. Studies indicated that the PEF protein apoptosis-linked gene 2 (ALG-2) binds Sec31A at ER-exit-sites (ERES) in a Ca2+-dependent manner, but in vitro and intact cell methodologies have been unable to adequately explain the specific role of ALG-2 and its PEF protein binding partner, peflin, in secretion. Chapter 2 describes experimental procedures, while Chapter 3 significantly expands upon the ALG-2- peflin regulatory machine. I find that although both proteins are fully dispensable for secretion, a peflin-ALG-2 heterocomplex binds to ERES via the ALG-2 subunit and confers a low, buffered transport rate, while peflin-lacking ALG-2 complexes can either enhance or inhibit transport of ER cargoes depending on expression level. This apparent bifurcated response indicates that PEF protein regulatory states may tune transport rates through expression level or Ca2+ changes, yet PEF proteins have never been observed regulating transport in response to Ca2+. In Chapter 4, I examine the roles of ALG-2 and peflin in response to physiological Ca2+-mobilizing agonists, and find that ALG-2 depresses ER export in epithelial NRK cells and enhances ER export in neuroendocrine PC12 cells. Furthermore, I find that Ca2+ signaling in the NRK cell model can produce opposing effects on secretion depending on signaling intensity and duration— phenomena that could contribute to cellular growth and intercellular communication following secretory increases or protection from excitotoxicity and infection following secretory decreases. Mechanistically, ALG-2-dependent depression of secretion involves decreased levels of the COPII outer shell and increased peflin targeting to ERES, while ALG-2-dependent enhancement of secretion involves increased COPII outer shell and decreased peflin at ERES. These data provide insights into how PEF protein dynamics affect secretion of important physiological cargoes such as collagen I and significantly impact ER stress.

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© Copyright 2021 John Robert Sargeant