PENTA EF-HAND PROTEIN ALG-2 REGULATES COPII ACTIVITY AT ERES IN RESPONSE TO CALCIUM SIGNALING

Author

Jacob Ryan Lapka, The University Of MontanaFollow

Year of Award

2025

Document Type

Thesis

Degree Type

Master of Science (MS)

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, Bruce Bowler

Keywords

PEF proteins, Protein Trafficking, ER-to-Golgi, Secretory Cargo, ER Exit Sites, ER Resident Proteins

Subject Categories

Cell Biology

Abstract

Intracellular protein trafficking is critical for maintaining cellular homeostasis, relying on highly regulated vesicular transport pathways originating from the endoplasmic reticulum (ER). Central to this process is the COPII coat complex, which governs cargo export at ER exit sites (ERES) by selectively packaging secretory proteins into transport vesicles. Recent studies highlight the regulatory role of calcium (Ca²⁺) signaling at ERES, where the Ca²⁺-binding Penta EF-hand proteins ALG-2 and peflin modulate COPII coat dynamics and cargo sorting in a context-dependent manner. In Chapter 2, we show that knockdown of the Inositol 1,4,5-trisphosphate receptor-3 (IP3R-3) in normal rat kidney (NRK) epithelial cells, which activates ALG-2 by increasing spontaneous cytosolic calcium oscillations, enhanced ER-to-Golgi transport of COPII client cargo. By examining trafficking at the earliest stages of ER-to-Golgi transport, we found that ALG-2 activation was linked to increased accumulation of client cargo at ERES, suggesting that ALG-2 activation modulates COPII early-stage cargo sorting under elevated cytosolic Ca²⁺ conditions. Chapter 3 utilizes live-cell imaging to reveal that transient Ca²⁺ signaling triggers a rapid but short-lived recruitment of ALG-2 and outer COPII coat components, Sec31A and Sec13, to ERES. In contrast, inner coat component Sec23A showed no change in localization, indicating that Ca²⁺-dependent modulation of coat assembly selectively influences outer COPII coat recruitment. The transient nature of outer coat recruitment suggests additional mechanisms that lead to the persistent changes in secretion detected long after ALG-2 activation. In Chapter 4, we explore a long-studied phenomenon, termed exodosis, in which ER resident proteins are secreted from cells treated with Ca²⁺-modulatory drugs, such as the Ca²⁺ ionophore A23187 or the SERCA pump inhibitor Thapsigargin (TG), for extended periods. We provide preliminary evidence that within NRK cells, ALG-2 and peflin act as gatekeepers in retention of ER resident proteins. Fluorescent microscopy-based assays demonstrate visual loss of endogenous ER resident proteins with short-term (1 hour) treatment of these drugs, however, experiments using brefeldin A (BFA) and MG132 revealed that the observed fluorescence loss did not align with classical secretion pathways or proteasomal degradation. Time did not allow a more complete exploration of these phenomena.

Recommended Citation

Lapka, Jacob Ryan, "PENTA EF-HAND PROTEIN ALG-2 REGULATES COPII ACTIVITY AT ERES IN RESPONSE TO CALCIUM SIGNALING" (2025). Graduate Student Theses, Dissertations, & Professional Papers. 12519.
https://scholarworks.umt.edu/etd/12519