29 Mar 2023
by Richa

Summary- paper 32: The K/HDEL receptor does not recycle but instead acts as a Golgi-gatekeeper

Jonas C. Alvim, Robert M. Bolt, Jing An, Yasuko Kamisugi, Andrew Cuming, Fernanda A. L. Silva-Alvim, Juan O. Concha, Luis L. P. daSilva, Meiyi Hu, Dominique Hirsz & Jurgen Denecke

Nature Communications, 2023

Questions/gaps addressed:

  • Golgi membrane-spanning sorting receptors mediate the sorting of cargo but also need to be sorted themselves.

  • KDEL receptor/ Erd2 is believed to be recycled between ER and Golgi, but those studies were based on C-terminal tagged Erd2. Does Erd2 really cycle between Golgi and ER?

Key methods:

  • Localization of AtErd2-YFP is mostly ER with some Golgi puncta, but the construct is not functional. YFP-TM-AtErd2 (YFP followed by an extra N terminal TMD) is mostly at the Golgi and is functional. Same localization dynamics when expressed the tagged At or HsErd2 constructs in human cells.

  • α-amylase-HDEL (Amy-HDEL) secretion assay: All Erd2 orthologs with greater than 50% similarity reduced secretion of Amy-HDEL. Same phenotype for untagged or YFP-TM-Erd2 construct but not the Erd2-YFP construct.

  • Mutation of a “cluster of lysines” near the ERD2 C-terminus proposed to form the retrieval motif for COPI coat for Golgi to ER transport, has no effect on function. Mutation of conserved leucines at position −3 and −5 from the C-terminus (note yeast Erd2 does not have L at -5) killed Erd2 function.

  • Disruption of LxLPA C terminal motif led to Erd2 redistribution to the ER that increased when more ligand was co-expressed. Why does LLGG mutant bind COPI to recycle back to ER instead of travelling further in the Golgi?

  • Tested other small tags at the C terminus of At or Hs Erd2: FLAG, c-myc, HA. FLAG and c-myc were non-functional and alos showed ER localization. HA tag was functional and localized to the Golgi.

  • High overexpression of WT but not ∆C5 Erd2 mutant, similar to BFA treatment, led to mixing of ER-Golgi and caused Arf1-RFP to become cytosolic, and prevented secretion of both Amy and Amy-HDEL. How high expression of Erd2 leads to complete collapse of the ER-Golgi system is unclear?

  • How does the low expression of Erd2 as compared to cargo proteins explain its function? If Erd2 is not packaged in the COPI retrograde carrier, what provides specificity to the cargo being packaged in the COPI vesicles?

Major takeaways:

  • YFP or RFP fused to the ERD2 C-terminus inactivate the receptor. Using YFP followed by an extra TMD (from ERP1 which is a PQ family protein but contains 8 TMDs) at the N-terminus seems to be the best tag of the combinations tested.

  • Exposure of a native C-terminus di-leucine motif (LXLPA) in Erd2 is important for its Golgi localization. How conserved is the dileucine motif? What does it bind to?

  • ERD2 cycles between ligand-bound and ligand-free configurations within the Golgi, but does not cycle between Golgi and the ER. How does Erd2 define the specifity of packaging of its cargos without being packaged itself in the COPI vesicle?