Predominant Golgi Residency of the Plant K/HDEL Receptor Is Essential for Its Function in Mediating ER Retention

Abstract

Accumulation of soluble proteins in the endoplasmic reticulum (ER) of plants is mediated by a receptor termed ER RETENTION DEFECTIVE2 (ERD2) or K/HDEL receptor. Using two gain-of-function assays and by complementing loss of function in , we discovered that compromising the lumenal N terminus or the cytosolic C terminus with fluorescent fusions abolishes its biological function and profoundly affects its subcellular localization. Based on the confirmed asymmetrical topology of ERD2, we engineered a new fluorescent ERD2 fusion protein that retains biological activity. Using this fusion, we show that ERD2 is exclusively detected at the Golgi apparatus, unlike nonfunctional C-terminal fusions, which also label the ER. Moreover, ERD2 is confined to early Golgi compartments and does not show ligand-induced redistribution to the ER. We show that the cytosolic C terminus of ERD2 plays a crucial role in its function. Two conserved leucine residues that do not correspond to any known targeting motifs for ER-Golgi trafficking were shown to be essential for both ERD2 Golgi residency and its ability to mediate ER retention of soluble ligands. The results suggest that anterograde ER to Golgi transport of ERD2 is either extremely fast, well in excess of the bulk flow rate, or that ERD2 does not recycle in the way originally proposed.

A signal motif retains Arabidopsis ER-α-mannosidase I in the cis-Golgi and prevents enhanced glycoprotein ERAD

“…4c). In contrast, the GFP-fused cis -Golgi membrane marker AtERD2, a recycling receptor for K/HDEL tetrapeptide-containing ER-resident proteins 42,43 , was fully absorbed into the ER upon BFA treatment (Fig. 4d).…”

“…5a) assuming that this would facilitate the Golgi-to-ER retrieval of MNS3. A similar approach has recently been used to retrieve HDEL-tagged STtmd-YFP (STtmd fused to yellow fluorescent protein) from the Golgi to the ER 43 . Both the wild-type and the HDEL-tagged MNS3 fusion proteins were transiently expressed in N .…”

“…Interesting is the importance of leucine residues in several different ER-Golgi localization signals. Only recently, a conserved di-leucine motif (LXL) near the cytoplasmic C-terminus of the plant K/HDEL receptor AtERD2 was described to be essential for its Golgi residency and biological function in mediating ER retention of soluble ligands 43 . Replacement of leucine residues resulted in a significant shift of AtERD2 to the ER.…”

Living on the edge: the role of Atgolgin‐84A at the plant ER–Golgi interface

“…Interestingly, the tubular extensions protruding from Golgi bodies and connecting individual Golgi bodies observed with Atgolgin‐84A fusions are very similar to tubules observed with a newly designed fluorescent fusions of the plant K/HDEL receptor (ERD2) that retains biological activity (Silva‐Alvim et al ., 2018). The ERD2 gene product was previously shown to exhibit a dual ER‐Golgi localization, but this was based on C‐terminal fusions, which have lost biological activity.…”

The biogenesis of CLEL peptides involves several processing events in consecutive compartments of the secretory pathway

“…ER retention by the KDEL-motif is mediated by Golgi-resident K/HDEL-receptors, which effect retrograde transport of soluble ER proteins from the Golgi back to the ER (Pelham, 1988; 11 Phillipson et al, 2001;Silva-Alvim et al, 2018). Cleavage by SBT6.1 may thus occur either in the ER or in the Golgi.…”