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
- 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?
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.
- 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.
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.