17 Feb 2023
by Richa

Summary- paper 18: Predominant Golgi Residency of the Plant K/HDEL Receptor Is Essential for Its Function in Mediating ER Retention

Fernanda A.L. Silva-Alvim, Jing An, Jonas C. Alvim, Ombretta Foresti, Alexandra Grippa, Alexandra Pelgrom, Thomas L. Adams, Chris Hawes, and Jurgen Denecke

The Plant Cell, 2018

Questions/gaps addressed:

  • How do the cascade of interactions between KDEL ligands, ERD2, G-proteins, and protein kinase A, explain the transport of K/HDEL proteins back to the ER?

Key methods:

  • Fusion protein Amy-HDEL (Barley α-amylase (Amy)) as cargo. Cotransfection with increasing amounts of ERD2a reduced the partial secretion of Amy-HDEL.

  • Used a hybrid/ fusion ERD2 construct (NbERD2ab)- N term and 4 TMs of Erd2a + 4 TM and C term of Erd2b. Fusion effective in retention of Amy-HDEL. The two forms are interchangeable.

  • Fluorescent reporters: ST-YFP or ST-YFP-HDEL. Agrobacterium tumefaciens-mediated transient expression in infiltrated tobacco leaf epidermis cells followed by confocal laser scanning microscopy. ST-YFP predominantly at the Golgi, ST-YFP-HDEL retained in the ER. Overexpression of Amy-HDEL caused a partial redistribution of ST-YFP-HDEL to the Golgi.

  • N- and C-terminal YFP tagged Erd2: Erd2-YFP at eR+ Golgi. YFP-Erd2 predominantly ER. Both defective in Amy-HDEL retention. Tagging kills Erd2 function. Addition of N-terminal signal peptide and a short decapeptide harboring an N-linked glycosylation site, secYFP-Erd2, is only at the Golgi but is non-functional.

  • Addition of an N-terminal transmembrane domain to Erd2, similar to the one in Erp1, (cytosolic YFP followed by an additional TMD) results in Golgi localization, and is partially functional for Amy-HDEL retention. Overexpression of Amy-HDEl does not result in ER re-localizatio of Erd2.

  • C-tail mutations:

    • dileucine in the C-tail: mutation kills Amy-HDEL retention. The mutant localizes to the ER + Golgi.
    • deletion mutant lacking the last predicted TM domain and the cytosolic tail of Erd2. Mutant in the ER.

Major takeaways:

  • Tetrapeptides KDEL and HDEL both prevent reporter protein secretion equally well in plant cells.

  • Both N- and C-terminal fluorescent tagging interferes with ERD2 activity.

  • N-terminal tagging of ERD2 can result in Golgi-localized fluorescent fusions as long as the ERD2 N terminus is lumenal. The lumenal N term needs to remain unobstructed for function.

  • Golgi residency and biological function require a conserved dileucine motif interrupted with a nonconserved amino acid (LXL) near the Erd2 C terminus.