The Biochemical and Structural Basis of Get3d’s Role in Photosynthesis

Author: Barlow, Alexandra Nichole

Year: 2025

Degree: Dissertation (Ph.D.)

Advisor: Clemons, William M.

Committee Members: Voorhees, Rebecca M.; Shan, Shu-ou; Newman, Dianne K.; Clemons, William M.

Option: Biochemistry and Molecular Biophysics

DOI: 10.7907/wht9-1974

Abstract

Tail-anchored (TA) membrane proteins, defined by a single C-terminal transmembrane domain, are inserted into the endoplasmic reticulum (ER) membrane via the guided entry of tail-anchored proteins pathway. The central targeting factor of this pathway is Get3, an ATPase that receives TA clients from upstream chaperones and mediates their delivery to the ER. Here, we identify and characterize a unique Get3 homolog, termed Get3d, distinguished by a C-terminal α-crystallin domain (αCD). We show that Get3d is conserved across plants and photosynthetic bacteria and demonstrate that it localizes to the chloroplast in plants. We present the X-ray crystal structure of Get3d, revealing unique features including the αCD and a client-binding chamber in the closed state. Biochemical analyses confirm that Get3d is an active ATPase capable of binding TA proteins in vitro. To investigate its physiological role, we identified the plant-like Get3d homolog in Synechocystis sp. PCC 6803 and generated deletion and complementation strains. Loss of Get3d impairs cell growth and pigment production, and proteomic analyses reveal widespread dysregulation, including up-regulation of transcriptional regulators and down-regulation of redox-associated proteins—suggesting a role in redox homeostasis. Complementation studies show that ATPase activity is necessary for restoring the expression of key photosynthesis-related proteins, while the αCD is critical for maintaining Get3d protein stability in vivo. Finally, co-immunoprecipitation coupled to mass spectrometry identifies putative Get3d interaction partners enriched in membrane-associated and photosynthetic proteins. Together, these findings establish Get3d as a biochemically distinct and functionally essential member of the Get3 family, with a potential role in redox regulation and photosynthetic homeostasis in diverse photosynthetic organisms.

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