The K/HDEL receptor does not recycle, but instead acts as a Golgi-gatekeeper

doi:10.21203/rs.3.rs-1781070/v1

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The K/HDEL receptor does not recycle, but instead acts as a Golgi-gatekeeper

https://doi.org/10.21203/rs.3.rs-1781070/v1

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posted 14 Jul, 2022

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The K/HDEL receptor (ER retention defective 2 or ERD2) does not recycle between compartments when sorting ER chaperones, contrary to the favoured model. A conserved C-terminal di-leucine motif specifically prevents ERD2 Golgi-to-ER transport and is not required for ER export. The Golgi-retention mechanism strips Golgi-membranes of the GTPase ARF1 so that ERD2 avoids accompanying its ligands in retrograde transport. When this motif is deleted or masked, introducing a fast ER-to-Golgi export signal or an alternative cis-Golgi retention signal re-activates ERD2. Meanwhile, forcing retrograde transport renders the receptor non-functional. We have established an in vivo ligand/receptor ratio far greater than 100 to 1, and propose a gatekeeper model to explain how few receptors at the Golgi can prevent the secretion of highly abundant soluble ER proteins. The underlying mechanism is conserved across kingdoms and will yield valuable insight into Golgi-mediated cargo sorting and cisternal compartment maintenance.

The Golgi apparatus plays a central role in sending and receiving membrane carriers to and from organelles of the secretory pathway. Its evolutionary origin is unknown, and how it maintains its identity despite the constant influx and efflux of transport vesicles continues to fascinate and divide the field^1,2,3. Membrane-spanning sorting receptors are a particularly interesting class of proteins as they mediate the sorting of cargo but need to be sorted themselves.

A central dogma to describe receptor-mediated transport was derived from the principle of receptor-mediated endocytosis at the plasma membrane⁴. A prominent example is the mannose-6-phosphate (M6P) receptor which binds lysosomal proteins in the Golgi, moves in clathrin coated vesicles to early endosomes, followed by ligand-release and receptor recycling back to the Golgi^5,6. Continuous recycling⁷ permits few receptors to sort many lysosomal proteins, and this was shown to be essential for vacuolar sorting in yeasts⁸ and plants⁹. Well-defined receptor mutants with defects in either anterograde or retrograde transport strongly support the recycling model^10,11.

Evidence that a similar recycling principle operates for highly abundant soluble ER residents bearing the KDEL or HDEL retention signal first arose from the detection of post-translational modifications that are Golgi-specific^12,13. An elegant genetic screen led to the identification of the K/HDEL receptor (ERD2) in yeast¹⁴, followed by the isolation of the human homologue¹⁵. Ligand-overproduction caused human ERD2 redistribution from the Golgi to the ER¹⁶, and the receptor-recycling principle was generally accepted by the field. Since detection of endogenous ERD2 proved technically difficult^17,18, the field used C-terminal ERD2 fusions for subcellular localisation and the demonstration of ligand-induced ERD2 redistribution to monitor its activity indirectly^16,19−22.

Having recently shown that fluorescent proteins such as YFP or RFP fused to the ERD2 C-terminus inactivate the receptor, we constructed a new fluorescent fusion (YFP-TM-ERD2) in which the ERD2 core remains unobstructed²³. This fusion was only detected at the Golgi apparatus even when ligands were overproduced. Both the Golgi residency and biological activity of ERD2 were found to be strictly dependent on a native C-terminus harbouring a novel di-leucine motif (LXLPA)²³. We concluded that this signal either mediates extremely fast “frequent flyer” ER export of ERD2 to explain the steady state levels at the Golgi, or that ERD2 may not recycle as currently believed.

Here, we provide a systematic set of experiments that strongly reject a “frequent flyer” model, and show that a Golgi-retention mechanism averts retrograde receptor transport to the ER. We discuss the implications for understanding Golgi-identity and how sorting machinery segregates from cargo.

YFP-TM-ERD2 can replace endogenous ERD2 in Nicotiana benthamiana and Physcomitrium patens

To obtain genetic validation for the new fluorescent fusion²³, we tried to generate stable Nicotiana benthamiana transformants co-expressing an ERD2ab antisense construct in a T-DNA together with either YFP-TM-ERD2 or ST-YFP-HDEL as control (Figure 1A). The frequency of control callus-formation was very low; shoots revealed the typical ER network for ST-YFP-HDEL (Figure 1B, panel 1) but failed to form roots. All control lines were subsequently lost, confirming that ERD2 knockdown is lethal²⁴. In sharp contrast, co-expressed YFP-TM-ERD2 resulted in fertile plants, showing punctate Golgi-structures (Figure 1B, panel 2). Seeds also produced fertile plants, and root cortex cells displayed typical Golgi structures (Figure 1B, panel 3). YFP-TM-ERD2 did not reveal any detectable ER network, even at the highest detector gain and contrast.

Further validation was also provided in the model bryophyte Physcomitrium patens using targeted “knock-in” of YFP-TM, followed by complete deletion of the second ERD2 gene (Figures 1C,D). The resulting moss-line showed normal growth expressing YFP-TM-ERD2 in punctate structures in the growing tips and newly formed cell plates (Figure 1E, panel 1, white stars). Expression was very low and high detector gain settings were needed, therefore also revealing autofluorescence of chloroplasts (Chl.). No structures reminiscent of ER were observed (Figure 1E, panel 2, white arrow heads). The results establish that a single YFP-TM-ERD2 gene expressed under its native promoter can functionally replace both endogenous ERD2 genes in P. patens.

Golgi residency of ERD2 is strictly linked to functionality in eukaryotes

To rule out that ERD2 Golgi residency is a plant-specific feature, ERD2 orthologs from 12 further eukaryotic organisms were tested in our functional assay²³. Figure 2A shows that the vast majority of ERD2 orthologs increased Amy-HDEL or Amy-KDEL retention in the cells comparable to Arabidopsis thaliana ERD2 (AtERD2 - second lane). Below a threshold of 50% homology with AtERD2, Amy-HDEL secretion was only weakly reduced (T. brucei) or not reduced at all (K. lactis, S. cerevisiae). S. cerevisiae ERD2 (ScERD2 – last lane) consistently induced AmyHDEL secretion, suggesting a dominant-negative effect on endogenous machinery.

Human ERD2 (HsERD2) mediates a very similar ER retention activity as AtERD2, as illustrated by a dose-response assay (Supplemental figure 3A), and shares the same sensitivity to C-terminal fusions as plant ERD2²³. YFP-TM-HsERD2 was found exclusively in the Golgi whilst the C-terminal fusion displayed a dual ER-Golgi localisation (Figure 2B). The former promoted strong Amy-HDEL retention whilst the latter did not (Figure 2C).

An earlier study of the human ERD2 C-terminus proposed that Serine209 controls ARF-GAP recruitment via PKA phosphorylation²¹. In our activity assay, neither the inactive (S209A) nor the phosphomimetic (S209D) mutation affected ERD2 function and the residue is not conserved between plant and human ERD2 (Figure 2D). More recently, a “cluster of lysines” near the ERD2 C-terminus was proposed to form an inducible COPI-retrieval motif²². Here we show that mutating each lysine (K206, K207) or both together does not alter the biological activity of human ERD2 (Figure 2C, Supplemental figure 3B). Instead, the conserved leucines at position -3 and -5 from the C-terminus (Figure 2D) are essential for human ERD2 activity (Figure 2C, last lane). A strict Golgi-localisation of human ERD2 mutants with wild type amy-HDEL retention activity was observed (Figure 2E) whilst additional labelling of the ER network was only seen when the leucines were mutated (LLGG).

To test if the ERD2-C-terminus can tolerate small epitopes as for instance the c-myc epitope¹⁶, we tested 3 different tags on either plant or human ERD2 for activity and localisation (Figure 2F). FLAG- and c-myc-tagged ERD2 showed strongly reduced biological activity compared to untagged ERD2 (Figure 2G) and were localised to both the ER and the Golgi (Figure 2H). A dose-response assay illustrates how strongly those two tags affected the functionality of plant and human ERD2 (Supplemental figure 3C) although weak residual activity may suffice for yeast complementation¹⁴. Interestingly, fusion of the HA tag (YPYDVPDYA) had only a minor effect on either plant or human ERD2 activity and resulted in predominant Golgi localisation (Figure 2H). This exception highlights the temperamental nature of C-terminal modifications, but also shows the strict correlation between Golgi residency and strong biological activity.

The C-terminal di-leucine motif of ERD2 prevents its recycling to the ER

To test if the di-leucine motif is a fast ER to Golgi export signal, fluorescence recovery after photobleaching (FRAP) was used to see if YFP-TM-ERD2 arrival at the Golgi is slowed down by mutating the signal. Figure 3A shows that the opposite is observed; the mutant recovers approximately twice as fast. Compared to the Golgi marker ST-YFP, the wild type ERD2 fusion behaves very similar and does not exhibit unusually fast “frequent-flyer” ER export to start with (Supplemental figures 4A, B). The results show that the di-leucine motif acts as a Golgi-retention signal, it prevents Golgi to ER retrograde transport rather than accelerating ER export. A faster Golgi-recovery of the mutant can be explained by the additional pool of ERD2 in the ER (Supplemental figure 4C) which supplements de novo synthesized ERD2 for more ER export.

To show that Ligand-induced ERD2 redistribution to the ER^20,22,25 could be caused by compromised Golgi-retention, we co-expressed secreted Amy or retained AmyHDEL together with either functional or non-functional ERD2-fusions (Figure 3B). YFP-TM-ERD2 shows no HDEL-mediated redistribution (Figure 3C). Deletion of the last 5 amino acids LQLPA (YFP-TM-ERD2ΔC5) results in partial ER localisation which is exacerbated upon HDEL-overdose (Figure 3D). The same HDEL ligand-induced redistribution from Golgi to ER can be seen with ERD2-YFP (Figure 3E). Interestingly, low expression of ERD2-YFP alone also favours an ER-localisation (Figure 3F, Supplemental Figure 5A). ERD2-YFP failed to yield rooted transgenic plants when co-expressed with ERD2ab antisense construct and weak ERD2-YFP fluorescence was mainly detected in the ER (Supplemental Figure 5B panels b3, b4). As the ERD2ab antisense was expressed from the same T-DNA, high expressing lines did not even reach the shoot stage.

The results show that ligand-mediated ER redistribution of ERD2 is simply caused by masking the di-leucine motif. Interestingly, ERD2 leakage to post-Golgi organelles remains undetectable in all conditions, suggesting that an additional mechanism prevents post-Golgi transport.

The Golgi-retention motif is required to inhibit COPI-mediated recycling.

It has been reported that ERD2 can cause mixing of Golgi and ER membranes²⁶ by recruiting ARF1-GAP to the Golgi apparatus²⁷. We therefore tested the influence of wild type and mutant ERD2 on the localisation of the GTPase ARF1. ARF1 typically colocalises with the Golgi-marker ST-RFP and post-Golgi structures (Figure 4A, white arrowheads), but when co-expressed with ERD2, ST-RFP redistributes to an ER-like super-compartment reminiscent of Brefeldin A treatment¹⁹ and ARF1 redistributes to the cytosol. ERD2 inhibits Amy-HDEL secretion at low expression levels without affecting constitutive Amy secretion²³ but at saturating expression levels Amy secretion is also inhibited (Figure 4B). The deletion mutant ERD2ΔC5 affects neither Amy-HDEL retention nor Amy secretion. The results strongly suggest that ERD2 inhibits COPI-mediated transport, and that the Golgi-retention motif is required for this. Although ERD2 has been reported to interact with ARF1 this was based on inactive C-terminal fusions²⁵.

COPII/COPI-mediated recycling of ERD2 is incompatible with its biological function

To test COPI-dependence more directly, we created an ERD2 hybrid (Figure 4C) by replacing the 9 most C-terminal amino acids with those of a P24 family member (p24δ5, AT1G21900), harbouring the di-hydrophobic sequence (YF) for COPII-mediated ER export and a canonical di-lysine motif (KKXX) for COPI-mediated recycling^28,29.

The hybrid showed no biological activity on HDEL cargo, but mutating the two lysines into serines (KKSS) induced receptor activity again (Figure 4D). CLSM analysis revealed complete ER retention of RFP-TM-ERD2::P24tail, with no detection in the Golgi bodies (Figure 4E, white arrow heads), illustrating the dominant nature of the COPI sorting signal. Destroying the COPI signal by replacing the two lysines with serines establishes predominant Golgi-residency with only traces detectable in the ER (Figure 4E).

Canonical COPI transport is clearly incompatible with ERD2 function, but the proposed COPII ER export signal in the p24 C-terminus²⁹ seems to compensate for the lack of the di-leucine Golgi retention signal.

An alternative Golgi-retention signal can re-activate ERD2-YFP

To test if the di-leucine motif can be replaced by an alternative Golgi localisation signal, of ERD2, we chose the newly identified N-terminal cytosolic Golgi retention motif (LPYS) of Arabidopsis thaliana α-mannosidase I (MNS3) that mediates cis-Golgi localisation³⁰. We first show that YFP-TM-ERD2 colocalises better with MNS3-RFP than with ST-RFP (Figure 6A,B). We supplemented inactive ERD2-YFP with the same N-terminal TM domain as in previous constructs (TM-ERD2-YFP) and then introduced the MNS3 N-terminus harbouring the LPYS signal (LPYS-TM-ERD2-YFP). The Amy-HDEL cargo sorting assay (Figure 6C) confirms that TM-ERD2 has almost wild type activity²³. TM-ERD2-YFP has lost this activity due to the devastating effect of C-terminally fused YFP. However, LPYS-TM-ERD2-YFP was partially re-activated despite the masked C-terminus. TM-ERD2-YFP remains partially ER-localised whilst LPYS-TM-ERD2-YFP is exclusively detected in the Golgi bodies (Figure 6D).

The results provide strong evidence that Golgi-retention per-se promotes ERD2 function, directly arguing against the receptor recycling model.

ERD2 mediates extra-stoichiometric retention of HDEL proteins

To establish relative numbers of ectopically expressed ERD2 versus Amy-HDEL proteins in vivo, we used quantitative immunoprecipitation with excess antibodies after 8 hours of continuous metabolic ³⁵S labelling. HA-tagged ERD2 had the highest biological activity of all tagged ERD2 variants (Figure 2G), therefore we expressed this receptor and Amy-HDEL from two separate GUS reference vectors to ensure comparable transfection efficiencies.

Despite equalised plasmid transfection, ERD2-HA produces a weak signal which is dwarfed by the high levels of cellular and secreted Amy-HDEL (Figure 6A). Nevertheless, ERD2-HA co-expression reduced secreted Amy-HDEL levels, accompanied by an increase in the cells. Since exactly two thirds of either cysteine or methionine are found in ERD2 when compared to Amy-HDEL (Figure 6B), the relative number of molecules could be determined by phosphor imaging and multiplying ERD2 values by a factor 1.5. This shows that there are approximately 4.5 additional Amy-HDEL molecules recovered in the cells for each ERD2 molecule introduced (Figure 6C).

A dose-response experiment with the longer standard 24 hours incubation was carried out using the same transfection conditions for Amy-HDEL and ERD2-HA as in Figure 6A to establish a baseline (Figure 6D, first two lanes). ERD2 plasmid was then progressively diluted up to 100-fold (Figure 6D, all further lanes) whilst the Amy-HDEL plasmid concentration was kept constant. Maximal Amy-HDEL retention was sustained up to 20-fold dilution of the ERD2-HA plasmid. 50-fold and 100-fold dilutions showed only a weak reduction in Amy-HDEL retention. We cannot detect in vivo labelled ERD2 under these conditions, but the internal marker GUS illustrates the quantitative nature of the dose-response assay (Supplemental figure 6A,B).

From these data we can ascertain that one introduced ERD2 molecule can prevent the secretion of well over 100 Amy-HDEL proteins. The true extra-stoichiometric retention capacity is likely to be much higher, because plasmid co-transfection is never 100% complete and ERD2-HA is slightly less active than wild type ERD2. Finally, and most importantly, a protein can only be secreted once, but retention requires endless recycling, as discussed below.

The recycling principle for protein sorting receptors has been popular in the field because it is thought to explain how few receptors can transport multiple ligands^7,9,31. However, whilst vacuolar/lysosomal proteins are sorted only once from secreted proteins, ER residents recycle back to the ER¹², the place where they were originally synthesized. Their perpetual escape to the Golgi would cause an endless “Sisyphus” task for ERD2. In addition, newly synthesized ER residents would add to the burden at each cycle. Given the high abundance of soluble ER residents³², such odds would defeat even the most efficient receptor, and this conceptual problem has not been widely considered to date. If ERD2 accompanies its ligands to the ER in COPI vesicles, it would need to return to the Golgi as a “frequent-flyer”. Here we have presented a systematic series of results that strongly argue against the frequent flyer recycling principle.

Firstly, lysine residues proposed as an inducible KKXX-like signal for COPI transport²² were completely irrelevant for ERD2-function (Fig. 2C), and they deviate from the strictly C-terminal position³³. Secondly, equipping the ERD2 C-terminus with well-established COPII and COPI transport signals^28,29 causes ER localisation and inactivity (Fig. 4), whilst mutating the canonical KKXX motif restores Golgi-localisation and activity.

Two further experiments provide strong arguments for Golgi-retention of ERD2. FRAP analysis shows that the di-leucine motif specifically slows down ERD2 turnover at the Golgi (Fig. 3). The signal is conserved in human ERD2 and promotes both activity (Fig. 2C) and Golgi-residency (Fig. 2E). More importantly, an alternative cis-Golgi retention signal³⁰ can restore ERD2 function when its di-leucine motif is masked by YFP (Fig. 6). Finally, ligand-induced redistribution of ERD2 fusions to the ER only occurs when the di-leucine motif is masked or deleted (Fig. 3D,E). This explains previously published data^16,20,22,25 and shows that the discrepancy does not lie with what is observed, but with what is deemed biologically relevant.

It is interesting that masking, mutating or even deleting the di-leucine motif did not lead to ERD2-leakage to post-Golgi compartments. ERD2 may thus contain 2 separate Golgi retention signals, the di-leucine motif to prevent ERD2 retrograde transport and a second to prevent anterograde Golgi-export. The latter remains a mystery³⁴, as does a full understanding of the mechanisms that maintain the spatial polar organisation of the Golgi stacks themselves³.

Our measurement of the in vivo ERD2-ligand stoichiometry suggests a ratio higher than 1:100. How can ERD2 avoid the “Sisyphus” paradox? Colloquially, the classic “frequent flyer” model can be illustrated by a taxi-driver who can take only a limited number of passengers. The driver can return to the collection point for another shuttle service, but whilst in transit any additional passengers have to wait. Our results suggest that ERD2 acts more like a “gatekeeper” at the cis-Golgi, illustrated by a bouncer who denies entry. Unlike a taxi driver, the bouncer holds its position at the gate and is able to repel a far greater crowd of individuals, including those making repeated attempts. This explains why the Golgi retention motif is crucial for ERD2-function and conserved in eukaryotes (Fig. 2A).

How could ERD2 avoid joining its ligands and maintain its gatekeeper position? We could show that the GTPase ARF1 is stripped from Golgi-membranes and redistributes to the cytosol when ERD2 is overexpressed (Fig. 4A). This is accompanied by inhibition of constitutive secretion (Fig. 4B) and this effect is dependent on the presence of the di-leucine motif for ERD2 Golgi-retention (Fig. 4B). HDEL cargo can be detected in the cis-Golgi³⁵ and may represent cargo transiently associated with ERD2. ARF1-GAP recruitment²⁷ may help to retain ERD2 in a COPI-free zone and stimulate/drive ligand-release into an adjacent COPI-coated subdomain.

Permanent Golgi-residency requires only minimal turn-over of ERD2, which explains why its expression under control of its own promoter in P. patens is extremely low (Fig. 1E) and its ER-to-Golgi transport is not faster than that of a typical Golgi marker (Fig. 3A). The gatekeeper model for ERD2 stands apart from the recycling receptors controlling post-Golgi trafficking routes^4,5,6,11 and inspires further thoughts on the unknown origins of the ER and Golgi. When COPI was first associated with retrograde rather than anterograde transport^36,37 the cisternal progression model for Golgi polarity gained momentum³⁸. Our present results support the idea that the plant cis-Golgi could be a more permanent core-structure³⁹ and corresponds to the mammalian ERGIC⁴⁰.

A precedent for a sorting facilitator that does not enter the vesicle carrier itself is Tango1, promoting collagen loading for ER export whilst effectively remaining behind in the ER membrane^41,42,43. Collagen is one of the most abundant secretory cargos in fibroblasts, and a “taxi-driver” mechanism could be easily overwhelmed. An example of a Golgi-resident sorting component is RER1⁴⁴, which also depends on its C-terminus to accumulate in the Golgi apparatus, but it controls accumulation of membrane proteins in the ER. The similarity of ERD2 with sweet transporters²², which are known to have multiple conformational states^45,46,47, may provide new clues to identify ancient transport mechanisms that could have initiated the formation of an ER-Golgi interface.

Future work should be devoted to experiments exploring 1) how ERD2 maintains its position at the Golgi apparatus without leaking beyond, 2) how it loads K/HDEL cargo for retrograde transport without joining and 3) how anterograde transport of non-ER proteins is achieved whilst the cis-Golgi retains its identity.

Recombinant DNA construction

All plasmid constructs were created via standard techniques including PCR amplification, overlap PCR, QuickChange PCR mutagenesis, gene synthesis, oligonucleotide annealing, restriction digests, gel purification and ligation and the E.coli strain for plasmid replication was strain MC1061⁴⁸. Supplementary table 1 lists all plasmids and constructs used from earlier work²³ and new derivatives described below

Double vectors for stable transgenic ERD2 anti-sense lines

For generation of transgenic N. benthamiana lines, a nbERD2AB-antisense fragment followed by 3’nos polyadenylation signal was extracted from pJCA60²³ as a NcoI-HindIII, sub-cloned together with a second fragment (BamHI-NcoI, harbouring a 3’ocs polyadenylation signal followed by the CaM35S promoter), into Agrobacterium tumefaciens plant expression vectors pTJA15, pTFLA32 and pTMY1 between BamHI-HindIII. NbERD2AB-antisense mRNA is then transcribed from the strong constitutive CaM35S promoter and the second cassette encodes either ST-YFP-HDEL (pTJCA85), YFP-TM-ERD2b (pTJCA86) or ERD2b-YFP (pTRB29) under the control of weaker TR2 promoter.

Targeted mutagenesis in P. patens

Establishment of the YFP-TM-PpERD2B-1/ΔPpERD2B-2 line

For construction of pYFP-TM-ERD2B-1, a fragment containing the YFP-TM sequence from pFLA30²³ was inserted directly between the end of the ERD2B-1 5’-UTR and the start codon of the ERD2B polypeptide coding sequence by overlap PCR, using primers (p1+p2+p3+p4, Supplementary Fig. S1; Supplementary Table 2). The product, containing 1120bp upstream of the coding region and 803bp genomic DNA commencing from the start codon was cloned into an EcoRV site of pBluescript II KS^-. For marker-free knock-in transformation of P. patens, 15μg of PCR amplified fragment (primers p5 and p6, Figure 1; Supplementary Fig. S1; Supplementary Table 2) of pYFP-TM-ERD2B-1 containing 1056bp and 760bp of 5’ and 3’ flanking fragments, respectively, was mixed with 1μg of supercoiled pMBL5 (GenBank Accession No. DQ228130) and used to transform P. patens by protoplast-PEG transformation⁴⁹. Primary transformants grown on G418 containing medium were transferred to non-selective medium and initially screened for correct 3’-end targeting by PCR. Loss of the circular selection plasmid was confirmed by sensitivity to G418. The selected transformants were further screened for 5’-end targeting and concatenation events by PCR followed by Southern hybridisation to establish a correctly targeted, single-copy plant. Genomic DNA (2.5μg) was digested with HindIII for electrophoresis and blotting onto nylon membrane. The probe comprising the 3’-end of YFP and the entire 3’-targeting fragment within YFP-TM-PpERD2B-1 (Supplementary Fig. S1, shaded box, primers p4 and p8, Supplementary Table 2) was labelled by PCR using 30% dTTP substituted with DIG-dUTP. The hybridisation and DIG detection was carried out in accordance with manufacturer’s instruction.

To create an ERD2B-2 knock-out plant (Figure 1; Supplementary Fig. S2; Supplementary Table 2), 5’- (956bp: primers p9 and p10) and 3’- (935bp: primers p11 and p12) targeting fragments were amplified from the genomic DNA located outside the ERD2B-2 coding region. These fragments were cloned either side of the 35S promoter driven nptII cassette in pMBL5DLΔS⁵⁰. The transgene containing 817bp of 5’ and 868bp of 3’ targeting sequence were bulk-amplified by PCR (primers p13 and p14) and used to transform P. patens::YFP-TM-ERD2B-1. The stable transformants were identified by two rounds of G418 selection. The targeted single-copy knock-out plants were confirmed by PCR followed by Southern hybridisation using an nptII-specific probe (primers p20 and p21). For the removal of the selection cassette, protoplasts of the YFP-TM-ERD2B-1/ERD2B-2KO plant were transiently transformed with 10μg of the supercoiled rice actin promoter-driven Cre recombinase plasmid and allowed to grow on protoplast regeneration medium without selection. After 2 weeks, individual regenerants were sub-cultured onto fresh standard medium with and without selection. The marker removal was confirmed by PCR testing antibiotic-sensitive colonies for the absence of the selection cassette.

ERD2 plasmids from different eukaryotic organisms

Gene synthesis services by Eurofins Genomics were used to obtain desirable coding sequences (CDS) and designed to be delivered in pUC57 vectors with restriction sites flanking both ends (ClaI and BamHI). Specific ERD2 sequences were selected from an open source online database (NCBI).

For the bioassay analysis all CDS mentioned above were sub-cloned into an existing pJA31 vector, a double expression vector with a GUS internal marker previously described^23,52, via classical cloning utilising ClaI and BamHI restriction sites, substituting ERD2b gene and finally yielding TR2:GUS-35s:oiERD2 (O. lucimarinus), TR2:GUS-35s:acERD2 (A. castellanii), TR2:GUS-35s:piERD2 (P. infestans), TR2:GUS-35s:ccERD2 (C. crispus), TR2:GUS-35s:gsERD2 (G. sulphuraria), TR2:GUS-35s:hsERD2 (Homo sapiens), TR2:GUS-35s:taERD2 (Tardigrade sp.), TR2:GUS-35s:tpERD2 (T. pseudonana), TR2:GUS-35s:pgERD2 (P. graminis), TR2:GUS-35s:klERD2 (K. lactis), TR2:GUS-35s:tbERD2 (T. brucei), TR2:GUS-35s:yERD2 (S. cerevisiae).

Human ERD2 derivatives

The construction of N-terminally tagged (YFP-TM-HsERD2) and C-terminally tagged (HsERD2-YFP) was carried out exactly as described previously for plant²³. Primer BglII-hERD2 was used to introduce a BglII site and a short linker (Ile-Ser) to the HsERD2 N-terminus. Primer HsERD2-NheI was used to introduce an NheI site and a short linker (Ala-Ser-Ala) to the huERD2 C-terminus. Both constructs were built and inserted either into the double expression vector with a GUS internal marker (pRB17 and pRB19) or into a T-DNA vector for Agrobacterium-mediated plant cell transformation for expression under the transcriptional control of the TR2 promoter (pTRB21 and pTJCA107).

Mutagenesis of human ERD2 was carried out using standard primers for quick-change mutagenesis as described in supplementary table 2 and implemented on the untagged human ERD2 construct in the dual expression vector for quantitative Amy-HDEL cell retention assays, followed by subcloning into the T-DNA vector encoding YFP-TM-huERD2 to study the mutants via CLSM analysis.

Epitope tagged plant and human ERD2

All C-terminal tags were inserted by trailer PCR using long antisense primers (Supplementary table 2) annealing either with plant or human ERD2, followed by the trailer harbouring the relevant epitope coding region, a stop codon and restriction site XbaI, for PCR amplification in conjunction with cool35S (5′-CACTATCCTTCGCAAGACC-3′) followed by ClaI-XbaI insertion into either the double expression vector with a GUS internal marker for cargo sorting assays (6 plasmids, see Supplementary table 1) or the T-DNA vector for CLSM analysis of the equivalent YFP-TM-ERD2 derivatives (6 further plasmids, see Supplementary table 1).

Deletions, hybrids and further derivatives of plant ERD2

To modify Arabidopsis thaliana ERD2b by deletions, chimeric hybrids and modified derivatives, a range of oligonucleotides were used either for direct trailer PCR or insertion of annealed primer pairs with sticky ends.

To replace the last 9 amino acids of ERD2 by the 9 amino acids of p24, the antisense primer ERD2b::p24tail was used combined with cool35S, followed by insertion into either the double expression vector with a GUS internal marker or the T-DNA vector for CLSM analysis. Mutagenesis of the di-lysine motif (KKSS) was done via Quick-change (Supplementary table 2), followed by the same subcloning reactions.

To test the influence of an alternative Golgi-retention signal (LPYS), the primers LPYSsense and LPYSanti were used to anneal a DNA fragment with NcoI and ClaI compatible sticky ends (encoding MSNSLPYSVKDVHYDNAKFRQR) to replace the YFP coding region in pFLA30²³ cut out by the same enzymes, resulting in the LPYS-TM-ERD2 coding region (pRB22). LPYS-TM-ERD2 and the control TM-ERD2 (pFLA33) were provided with a C-terminal YFP in the same way as described for ERD2 before²³, resulting in double expression vectors with a GUS internal marker plasmids pRB25 and pRB26, and the T-DNA vectors pTRB25 and pTRB26.

ERD2 constructs for re-distribution assays

To remove the di-leucine motif, the 5 last codons of ERD2b were removed by PCR amplification with primer ERD2b∆C5 anti, resulting in a coding region devoid of codons specifying the amino acids LQLPA, resulting in pTJCA88. The 35S-promoter driven ERD2b-YFP construct (pTJA10²³) was re-constructed by replacing the 35S promoter by the weaker TR2 promoter by EcoRI-ClaI substitution, yielding pTMY1. To test the influence of over-expressed Amy and Amy-HDEL on several fluorescent ERD2 derivatives, the corresponding genes were recovered by HindIII digestions from pAmy and pAmy-HDEL respectively, followed by blunting with Klenow and further digestion with EcoRI. Plant expression vectors pTJCA88, pTFLA32, and pTMY1 were prepared by SnaBI-EcoRI digests followed by dephosphorylation, resulting in the dual expression plant vector plasmids pTRB6, pTRB7, pTRB8, pTRB9, pTMY3, pTMY4 (supplemental table 1).

Further fluorescent markers

The Golgi marker ST-RFP under the transcriptional control of the weak TR2 promoter in plasmid pTJA37 was described previously²³. To generate an alternative Golgi-marker to specifically highlight cis-cisternae³⁰, the cytosolic N-terminus, the TM domain and a portion of the lumenal domain of MNS3 was amplified with MNS3 ClaI and MNS3 SalI from N. benthamiana protoplast cDNA, to replace the corresponding domains of ST-RFP²³, resulting in MNS3-RFP (pRB23). Subcloning into the T-DNA vector for Agrobacterium-mediated plant cell transformation for expression under the transcriptional control of the TR2 promoter resulted in pTRB23. ARF1-RFP was created by amplification of the ARF1 coding region as a NcoI-NheI fragment using primers ARF1-NcoI and ARF1-NheI and fused to an NheI-BamHI RFP fragment from pAW7 described earlier¹¹, to be expressed with the TR2 promoter.

Plant material and transient gene expression

Sterile grown N. benthamiana⁵² plants, protoplast preparation, electroporation and subsequent incubation and harvesting were done as described previously²³. The tobacco leaf infiltration procedure with soil grown plants was done as described too⁵³.

Confocal laser scanning microscopy

48 hours after infiltration, slides with tobacco leaf squares were prepared with tap water and imaged using an upright Zeiss LSM 880 Laser Scanning Microscope (Zeiss) with a PMT detector and a Plan-Apochromat 40x/1.4 oil DIC M27 objective using settings as described²³.

FRAP analysis

Samples to be used in fluorescence recovery after photobleaching (FRAP) studies were pre-treated to promote Golgi lock-down before analysis. 48 hours after infiltration, small sections of the infiltrated leaves were removed and kept in a solution containing latrunculin B solution to promote disruption of the cytoskeleton and stop Golgi movement. Infiltrated tobacco leaf squares (0.5 x 0.5 cm) were used for drug treatment preceding confocal imaging for photobleaching recovery studies, FRAP. To promote actin depolymerization and stop Golgi movement leaves were treated with latrunculin B⁵⁴. Samples were submerged in 12µM solution of the latrunculin B (Cayman Chemical Co.) in water for one hour and analysed soon after.

Samples were then analysed via confocal laser scanning microscopy (CSLM). Golgi bodies showing both RFP and YFP fluorescence were selected as region of interest (ROI) and either bleached (black arrow-head) or kept as a control (white arrow-head).

Zen 2.3 black edition (Zeiss) software was used to record pre- and post-bleached signals and to modulate laser beam intensity. Signals were sampled before bleach treatment using standard confocal setting as described before. Bleaching was achieved by scanning with high-intensity illumination of selected regions of interest (ROI) and every 30 seconds after bleaching with low-intensity illumination following recommendation of previously published protocol⁵⁴.

Correlation analysis

Post-acquisition image processing was performed with the Zen 2.3 lite blue edition (Zeiss) and ImageJ ((http://rsb.info.gov/ij/)). Image analysis was undertaken using the ImageJ analysis program and the PSC co-localization plug-in⁵⁵ to calculate co-localization and to produce scatter plots as described before¹¹.

Enzyme assays

Measurement of α-amylase activity and calculation of the secretion index (ratio of extracellular to intracellular enzyme activities) were done as described previously^23,51,56. For GUS-normalised effector dose-response assays, the GUS enzyme essay was used. To reach best transfection practice (BTP), new dual expression plasmid preparations were first subject to transfection quality control by measuring the GUS activity after standard electroporations as described earlier²³. However, plasmid dilutions were used to determine the point at which GUS activity starts to reach a plateau. A relative activity of 30 units (given in ΔOD per mL protoplast suspension and per hour enzyme incubation) prior to plateau conditions was deemed acceptable for BTP. Plasmids that reached the GUS activity plateau with lower than 30 units were deemed unsuitable and were discarded. For GUS-normalised comparisons of different effector plasmids and for dose-response assays, plasmid doses were strictly determined volumetrically as a percentage of the maximum dose resulting in 30 GUS units.

Generation of transgenic plants by leaf-disk transformation

N. benthamiana plants were obtained via Agrobacterium infection of leaf disks⁵⁷. Selection of transformants was accomplished in MS medium supplemented with 3% sucrose and containing 100 μg/mL kanamycin and 250 μg/mL cefotaxime. Regenerated plants were analysed and scored by CLSM.

In vivo labelling and immuno-precipitation

In vivo labelling of N. benthamiana protoplast suspensions was essentially done as described previously⁵⁸ with the following modifications: 4 repeats of standard electroporations²³ yielding 2.5ml protoplast suspensions each were pooled together for each sample (mock, Amy-HDEL and Amy-HDEL + ERD2-HA). After 1hour rest in a standard 9cm Petri Dish, the 10 mL pools were centrifuged at 100 rpm in conical tubes, followed by the removal of the dead cell pellet and the majority of the medium underneath the floating band of live cells. Protoplasts were then resuspended in a final volume of 2 mL TEX buffer and supplemented with 0.5 mL TEX containing 500 mCi/mL Pro-mix (70% 35S-methionine and 30% 35S-cysteine, Amersham Life Science), followed by incubation for 8 hours at room temperature in the dark and harvesting of washed cell pellets and clear culture medium using the standard procedure. Culture medium was kept on ice for further work. Lysis of washed cell pellets was carried out by resuspending the washed cell pellet with 950 microlitres of ice-cold homogenization buffer (200 mM Tris-Cl, pH 8.0, 300 mM NaCl, 1% Triton X-100, 1 mM EDTA, and 2 mM phenylmethylsulfonyl fluoride). The homogenate was then centrifuged for 5 min in a minicentrifuge, and the supernatant was kept on ice for further work.

Immunoprecipitations were either carried out with 500 microlitres of culture medium or 200 microlitres of the cell lysis fraction, each representing 20% of the total labelled protoplast suspension, allowing direct calculation of secretion indices after quantification of signals from medium and cells. All manipulations were performed on ice or at 4°C using ice-cold buffers. NET gel buffer (50 mM Tris-Cl, pH 7.5, 150 mM NaCl, 1 mM EDTA, 0.1% Nonidet P-40, and 0.02% NaN3 and supplemented with 0.25% gelatin) was used to bring either the medium or the cell lysis fraction up to 1mL. After centrifugation to remove any remaining debris, the supernatant was incubated on ice with an excess of either anti-HA antibody or anti-amylase antibodies for 1 hour, allowing for the complete precipitation of all ERD2 or Amy-HDEL proteins after addition of protein A-sepharose and subsequent washing steps as described⁵⁸. After the last wash, all liquid was removed from the washed protein A-sepharose pellets using a refined glass capillary. The pellets were then supplemented with thirty microlitres of SDS-PAGE loading buffer (200 mM Tris-Cl, pH 8.8, 5 mM EDTA, 1 M sucrose, 20 mM DTT, 2.5% SDS, 0.1% bromophenol blue) and the suspensions were incubated at 90°C for 5min. The sample was then centrifuged for 2 min in a minicentrifuge and 20 microlitres was separated on SDS-PAGE (10% strength), followed by electroblotting on nitrocellulose. Dried nitrocellulose sheets were analysed by phosphorimaging. Sample peak selection and detection were achieved by Aida version 4.14 and detector Fuji FLA-5000 respectively. Error bars are standard errors of three independent repeats. Arbitrary units of pixel intensity are compared relative to other values within the same experiment, and corrected for the 2/3 ratio of amino acids methionine and cysteine in ERD2 relative to Amylase.

Acknowledgments

The work in this article was in part supported by the European Union (projects HPRN-CT-2002-00262 and LSH-2002-1.2.5 − 2), the Biotechnology and Biological Sciences Research Council (BBSRC) project nr. BB/D016223/1 and The Leverhulme Trust (F/10 105/E). Jonas C. Alvim is grateful for a PhD scholarship awarded from the Conselho Nacional de Desenvolvimento Científico e Tecnológico – Brasil (CNPq 201192/2014-4). Robert M. Bolt is grateful for a Faculty of Biological Sciences PhD studentship funded by the University of Leeds. Reese’s Cups (Hershey Company, 1025 Reese Ave, Hershey, PA 17033) is thanked for providing food for thought. Jack Ranger and Nicoletta Bencka are thanked for contributing to the construction of recombinant plasmids (Supplementary table 1). David Gershlick is thanked for scientific discussion and critically reading the manuscript.

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The K/HDEL receptor does not recycle, but instead acts as a Golgi-gatekeeper

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