Fig 1.
Examples of starting tripeptides for several classes of secreted vertebrate proteins with conserved, ER-ESCAPE motifs.
All tripeptides found in NCBI Protein database searches (by gene name and BLASTP) are listed a single time for each protein, with vertebrate taxon notations in single-letter codes on the right. Signal peptide cleavage sites were predicted by SignalP 4.1 Server (http://www.cbs.dtu.dk/services/SignalP/) [6], Phobius (http://phobius.sbc.su.se/) [7], and/or experimental evidence noted on NCBI Proteins database. (See S1 Table for accession number, species name, and brief sequence of representative taxon for each tripeptide.) Color-coding based on relative contribution of each amino acid position to the strength of the ER-ESCAPE motif is as noted in Results and Discussion. AMBN, ameloblastin; AMELX, amelogenin, X-linked; AMTN, amelotin; BSP, bone sialoprotein; DMP1, dentin matrix acidic phosphoprotein 1; DSPP, dentin sialophosphoprotein; ENAM, enamelin; ER-ESCAPE motif, Endoplasmic Reticulum Exit by Soluble Cargo using Amino-terminal Peptide-Encoding motif; GH1, growth hormone 1; NCBI, National Center for Biotechnology Information; OPN, osteopontin; proMMP-9, pro-matrix metalloproteinase-9; PTH, parathyroid hormone.
Fig 2.
Composition of amino-terminal tripeptide is critical for ER trafficking of DSPP and AMELX in HEK293A cells.
(A) Substitution of amino acids 1 and/or 3 of IPV with other hydrophobic (alanine, phenylalanine, methionine, or tyrosine), polar/uncharged (serine or threonine), or positively charged (arginine) amino acids in DSPP’s ER-ESCAPE motif continued to support efficient trafficking. Immunoblot shows protein levels of wild-type (IPV) and modified-tripeptide DSPP intact protein as well as cleavage product, DSP, in cell lysate (“L”) and conditioned media (“M”). Antibody to DSP domain was used for detection of intact DSPP as well as DSP-related cleavage products in the media. (B) Retention of DSPP in the cell shows trafficking of DSPP was disrupted by replacement of proline with leucine, serine, or threonine or substitution of either hydrophobic position with an acidic amino acid (D or E). Inclusion of 6xFlag-tag in noted constructs caused expected higher Mr of intact DSPP protein. (C) Mutations in DSPP’s IPV motif do not activate IRE1 pathway of the UPR (or ER stress). Note that 52 hr of expression of DSPP starting with wild-type (IPV) or mutant (IPD) did not cause the IRE1 pathway’s XBP-1 splice event associated with UPR. Addition of tunicamycin (known to activate UPR by inhibiting addition of N-linked oligosaccharides) for 5 hr resulted in abundant expression of the shorter, UPR-activating spliced XBP-1 message. (D) Wild-type (MPL) AMELX with carboxy-terminal Myc-tag was well trafficked out of the cell. AMELX with replacement tripeptide lacking number 2 position, proline (ISV or FSM), or replacement of starting hydrophobic with an acidic amino acid (EPL) all failed to traffic out of the cells. Faint band at higher Mr is due to small amount of AMELX that acquired a N-linked oligosaccharide. Anti-Myc antibody was used for detection. Cells were collected 18 hr (DSPP) or 24 hr (AMELX) posttransfection. Six μg of cell lysate protein or 20% of concentrated media was used for western blot analyses. Detection was with LI-COR Odyssey using IR-labeled second antibodies. Numbers on left are molecular weight standards in kDa. AMELX, amelogenin, X-linked; APD, alanine-proline-aspartic acid; APV, alanine-proline-valine; DSP, dentin sialoprotein; DSPP, dentin sialophosphoprotein; EPL, glutamic acid-proline-leucine; EPV, glutamic acid-proline-valine; ER, endoplasmic reticulum; ER-ESCAPE motif, ER-Exit by Soluble Cargo using Amino-terminal Peptide-Encoding motif; FPM, phenylalanine-proline-methionine; FSM, phenylalanine-serine-methionine; HEK293A, human embryonic kidney cell line 293A; ILV, isoleucine-leucine-valine; IPD, isoleucine-proline-aspartic acid; IPR, isoleucine-proline-arginine; IPV, isoleucine-proline-valine; IR, infrared; IRE1, inositol-requiring enzyme 1; ISV, isoleucine-serine-valine; ITV, isoleucine-threonine-valine; MPL, methionine-proline-leucine; RPV, arginine-proline-valine; SPV, serine-proline-valine; TPV, threonine-proline-valine; UPR, unfolded protein response; XBP-1, X-box-binding protein 1; YPD, tyrosine-proline-aspartic acid; YPY, tyrosine-proline-tyrosine.
Fig 3.
Addition of N-linked oligosaccharide at fourth position does not change efficiency of amino-terminal tripeptide-enhanced GH trafficking.
(A) Chemical structure of N-linked glycosylation on APVNTT peptide (amino-terminal alanine at top). (B) Western blot: GH starting with high-efficiency motif, APV, obtained lower steady-state levels in HEK293A cells compared to same protein starting with predicted nonbinding motif, EET. Addition of N-linked oligosaccharide (NTT motif inserted at position 4) caused PNGaseF-susceptible increases in size (Mr) but did not change relative trafficking efficiencies of either strong (APV-NTT) or nonbinding (EET-NTT) proteins. The oligosaccharides on cell layer–associated GH by both constructs were removed by Endo H digestion, showing no Golgi modifications. Cells and media were collected 16 hr posttransfection. Three μg of cell lysate or 6% of concentrated media was used for western blot analysis with goat anti-human GH. LI-COR IR-fluorescent second antibodies were used for detection on LI-COR’s Odyssey scanner. Numbers on left are molecular weight standards in kDa. APV, alanine-proline-valine; EET, glutamic acid–glutamic acid–threonine; GH, growth hormone; HEK293A, human embryonic kidney cell line 293A; IR, infrared; NTT, asparagine-threonine-threonine.
Fig 4.
Erv29p is critical for trafficking of DSPP and PRY-family proteins in S. cerevisiae.
(A) Immunoblot of wild-type DSPPFlag (IPV) or DSPPFlag starting with the mutant tripeptide motif (IPD) in α or erv29Δ cell lysates. Yeast α or erv29Δ cells were transformed with pYES expression plasmid encoding IPV- or IPD-DSPPFlag under GAL1 promoter and induced for 5 hr. For rescue experiments, pAG425GPD plasmid constitutively expressing Erv29p was cotransformed with DSPP-encoding plasmids. Note that IPV-DSPPFlag was well trafficked out of the ER/cells of wild-type α cells, while IPD-DSPPFlag protein was retained. The erv29Δ cells could not efficiently traffic IPV-DSPPFlag protein unless it was cotransformed with Erv29p-expressing plasmid (rescue). Media contained Kex2-digested, Flag-tagged carboxy-terminal fragment DPP. Four μg of cell lysate protein and 10% of the concentrated conditioned media were used for Flag-tag detection on western blots. (B) Yeast wild-type α cells or erv29Δ cells were transformed with pYES expression plasmids encoding Pry1p or Pry2p, each with 2xHA-tags near the amino-terminus. Five hr after induction, 30 μg of cell lysate protein was analyzed by western blots. For each construct, higher Mr bands (solid red arrowheads) were proteins containing Golgi-acquired posttranslational modifications, while ER-retained proteins lack these modifications and electrophorese several kDa faster (open green arrowheads). Note both APA/APV and APD showed increases in the smaller ER forms in erv29Δ cells, while only APD shows abundance in ER forms in wild-type (α) cells. (C) Pry1p and Pry2p modified to express both 2xHA-tag (near amino-terminus) and Myc (carboxy-terminus) showed faster electrophoresing proteins (ER) were not due to endogenous protease activity. LI-COR IR-fluorescent second antibodies were used for detection on LI-COR’s Odyssey scanner. Numbers on left are molecular weight standards in kDa. APA, alanine-proline-alanine; APD, alanine-proline-aspartic acid; APV, alanine-proline-valine; DPP, dentin phosphoprotein; DSPP, dentin sialophosphoprotein; ER, endoplasmic reticulum; Erv29p, ER-derived vesicles protein 29; HA, hemagglutinin; IPD, isoleucine-proline-aspartic acid; IPV, isoleucine-proline-valine; IR, infrared; Kex2, kexin 2; PRY, pathogen-related yeast.
Fig 5.
Immunofluorescence microscopy of HEK293A cells shows Surf4 accumulates in and around ERESs.
(A) Fluorescent signal for endogenous Surf4 (green) was strongest at punctate structures positive for ERES marker Sec23 (red). Note additional Surf4 fluorescence in weblike structures surrounding ERES. (B) HA-Surf4 signal (green) was observed within the ERGIC (ERGIC-53, red). (C) HA-Surf4 (green) showed only low levels of colocalization with cis-Golgi marker, giantin (red). (D) Mutation of proposed COPI recycling motif by replacement of two of three near-carboxy-terminal lysines to alanines (HA-Surf4-AAK, green) increased colocalization with cis-Golgi marker, giantin (red). (E) Newly synthesized HA-Surf4 (green) was found at low levels in the rER (Sec61 marker, red). (F) Surf4 (green) did not colocalize with chaperone, calnexin (red), in the quality control domain. HEK293A cells were transfected with wild-type HA-tagged Surf4 plasmid (B, C, E), or Surf4KO HEK293A cells were transfected with carboxyl-terminal di-lysine mutation, HA-Surf4-AAK (D), 18 hr prior to fixation. Bars = 5μm. The cells in each panel are shown 3 times, first with organelle marker, then Surf4 and final panel (with magnified insert) showing overlap. Alexa Fluor secondary antibodies were used for detection. Images were obtained using an LSM 780 (Carl Zeiss) confocal microscope (488 and 561 nm excitation lines; 500–560 and 600–660 nm capture) and Zeiss Axio Imager Z1 with Apotome 2 (single Z stack slice). Images were analyzed using Zeiss Zen software. AAK, alanine-alanine-lysine; COP, coat protein complex I; ERES, endoplasmic reticulum exit site; ERGIC, endoplasmic reticulum–Golgi intermediate compartment; HA, hemagglutinin; HEK293A, human embryonic kidney cell line 293A; rER, rough ER; Surf4, surfeit locus protein 4.
Fig 6.
Surf4 is essential for keeping AMELX, DSPP, and GH at low concentrations within ER.
(A) CRISPR/Cas9 technology was used to delete SURF4 alleles from HEK293A cells (Surf4KO). Endogenous Surf4 was detected in HEK293A cell lysate (left lane) but not in Surf4KO cell lysate (center lane). Right lane shows reexpression of Surf4 using plasmid-encoding HA-tagged Surf4 in Surf4KO. Introduction of HA-tag slightly increased the Mr of Surf4. Detection of Surf4 was with affinity-purified rabbit antibody to carboxy-terminal peptide. Lower panel: Detection of β-Actin serves as loading/protease controls. (B) Trafficking of secreted proteins lacking Surf4-binding motifs were unaffected by loss of Surf4. SEAP and LPO-Gluc were equally well secreted from normal and Surf4KO cells. Conditioned media were harvested 22 hr posttransfection. SEAP secretion was assayed with 5μl of conditioned media using QUANTI-Blue kit. Luciferase activity was determined using 5 μl of conditioned media with BioLux Gaussia Luciferase Assay kit following Assay Protocol II. (Error bars are SEM with a transfection sample size of n = 5 [SEAP] and n = 6 [LPO-Gluc]) (C) AMELXmyc starting with MPL (Lane 1) well trafficked out of wild-type cells, but mutant EPL-AMELX (Lane 2) was not. Neither protein was efficiently trafficked out of Surf4KO cells (Lanes 3 and 4). AMELX was detected using primary antibody to Myc-tag. (D) Trafficking of wild-type AMELX (MPL) in Surf4KO cells was rescued by coexpression of either HA-Surf4 (Lane 1) or yeast’s Erv29p (Lane 3), but trafficking of EPL-AMELX was not rescued by either cargo receptor (Lanes 2 and 4). Coexpression of Surf4 lacking proposed motif for COPI recycling to ER (HA-Surf4-AAK) also could not rescue trafficking of MPL-AMELX (Lane 5). (E) Trafficking of IPV-DSPP in HEK293A cells (Lane 1) was lost in Surf4KO cells (Lane 3). There was negligible trafficking of IPD-DSPP in either wild-type (Lane 2) or Surf4KO cells (Lane 4). (F) Coexpression of HA-Surf4 (Lane 1) or HA-Erv29p (Lane 3) rescued IPV-DSPP trafficking in Surf4KO cells but not for IPD-DSPP (Lanes 2 and 4). Primary antibody to mDSP domain was used to detect intact DSPP and its DSP fragment. (G) Evidence for aggregate formation by DSPP and AMELX (Myc-tagged) in Surf4KO cells. Top panel: Surf4KO cells expressing DSPP were briefly pelleted and then treated for 10 min with buffer containing digitonin (CEB) with (+) or without (-) 10 mM Ca2+ and pelleted at >100,000 x g. As observed on western blots, 10 mM Ca2+ stabilized a portion of DSPP in the pellet fraction. In the bottom panel, AMELX (Myc-tagged) formed stable aggregate in Surf4KO cells with most remaining in >100,000 x g pellet after solubilizing cells with an MEB for 10 min. (H) Surf4-trafficked cargo with motifs other than Φ-P-Φ. Trafficking of GH lacking one hydrophobic amino acid (FPT), serine replacing proline at position 2 (ISV), or both lacking the proline, plus replacement of one hydrophobic with a positive-charged amino acid (RSV) were all rescued in Surf4KO cells upon coexpression of HA-Surf4 protein. Trafficking of di-acidic EET-GH was not rescued by HA-Surf4. (I) LPO-Gluc, noted in Panel B as not using Surf4, acquired lower steady-state levels when wild-type motif QTT was replaced with strong ER-ESCAPE motifs (RSV or IPV). Same proteins expressed in Surf4KO cells retained their high steady-state levels. Cells were harvested 22 hr posttransfection. The Luciferase activity was normalized to total protein (Luciferase units/mg protein). Error bars are SEM with sample size of n = 6 and P < 0.001 (**). For above experiments, cells were collected 18 hr (DSPP and GH) or 24 hr (AMELX) posttransfection. Ten μg of cell lysate protein and 20% of concentrated medium were used for western blots of DSPP and AMELX. GH analyses used 3 μg of cell lysate protein and 6% of concentrated media. LI-COR IR-fluorescent second antibodies were used for detection on LI-COR’s Odyssey scanner. Numbers on left are molecular weight standards in kDa. Φ-P-Φ, hydrophobic-proline-hydrophobic; AAK, alanine-alanine-lysine; AMELX, amelogenin, X-linked; AMELXmyc, Myc-tagged human AMELX; Cas9, CRISPR-associated 9; CEB, Cytosol Extraction Buffer; COPI, coat protein complex I; CRISPR, clustered regularly interspaced short palindromic repeat; DSP, dentin sialoprotein; DSPP, dentin sialophosphoprotein; EET, glutamic acid–glutamic acid–threonine; EPL, glutamic acid-proline-leucine; ER, endoplasmic reticulum; ER-ESCAPE motif, ER-Exit by Soluble Cargo using Amino-terminal Peptide-Encoding motif; Erv29p, ER-derived vesicles protein 29; FPT, phenylalanine-proline-threonine; GH, growth hormone; HA, hemagglutinin; HEK293A, human embryonic kidney cell line 293; IPD, isoleucine-proline-aspartic acid; IPV, isoleucine-proline-valine; IR, infrared; ISV, isoleucine-serine-valine; LPO-Gluc human lactoperoxidase with carboxy-terminal luciferase; mDSP, mouse DSP; MEB, membrane extraction buffer; MPL, methionine-proline-leucine; N/S, not statistically significant; QTT, glutamine-threonine-threonine; RSV, arginine-serine-valine; SEAP, secreted alkaline phosphatase; Surf4, surfeit locus protein 4.
Fig 7.
Examples of starting tripeptides in soluble proteins that are predicted not to interact with Surf4/Erv29p.
After removal of leader sequences, amino-terminal tripeptides of TOP: vertebrate and fungal/yeast soluble ER-resident chaperone/modifying proteins and BOTTOM: vertebrate fibrillar collagens too large to fit in standard COPII exit vesicles. Each tripeptide found in NCBI Proteins database searches (by gene name or BLASTP) is listed with vertebrate taxon notations in single letter codes at right, indicating this tripeptide was found at least one time for this taxon (e.g., M = mammal). (See S2 Table for accession number, species name, and brief representative sequence.) Color-coding based on relative contribution of each amino acid position to strength of the ER-ESCAPE motif. CALR, calreticulin; COL1A1, collagen type 1 alpha 1; COL2A1, collagen type 2 alpha 1; COL3A1, collagen type 3 alpha 1; COL6A1, collagen type 6 alpha 1; COL1A2, collagen type 6 alpha 2; COL6A3, collagen type 6 alpha 3; COL7A1, collagen type 7 alpha 1; COPII, coat protein complex II; ER, endoplasmic reticulum; ER-ESCAPE motif, ER-Exit by Soluble Cargo using Amino-terminal Peptide-Encoding motif; ERO1, ER oxidoreductase 1; Erv29p, ER-derived vesicles protein 29; F-GRP78, fungal glucose-regulated protein 78; GRP78, glucose-regulated protein 78; GRP94, glucose-regulated protein 94; NCBI, National Center for Biotechnology Information; PDI, protein disulfide isomerase; PDIA2, PDI family A member 2; PDIA4, PDI family A member 4; Surf4, surfeit locus protein 4; TANGO 1, transport and Golgi organization 1.
Fig 8.
Relative affinities of ER-ESCAPE motifs for cargo receptor by analyses of GH steady-state levels.
(A) Pre-confluent cells were separately transfected with expression plasmids encoding human GH with 60 different ER-ESCAPE motifs as noted. Eighteen hr posttransfection, cells were washed, extracted with lysis buffer, and analyzed by GH ELISA. GH values (ng GH/mg protein) were normalized within each experiment to the amount of IPV-GH. Each histogram bar represents mean ± SEM. of at least three independent transfections for each construct. (Means of triplicate GH ELISA analyses were used for each extract). Larger error bars among poorest ER-ESCAPE motifs may reflect a variable amount of aggregate formation by larger amounts of accumulated GH. Note that Φ-P-Φ ER-ESCAPE motifs are among the most efficient at trafficking GH, while those including acidic amino acids or glutamines are much less effective. Substitution by positively charged amino acids (generally, R better than K) retained effective trafficking, while loss of proline in position 2 was otherwise detrimental. (B) Prediction of value that each amino acid in positions 1, 2, and 3 adds to the quality of the ER-ESCAPE motif binding to Surf4 based on combination of ELISA and database search results. Note that some combinations may give results not predicted by simply summing a tripeptide’s three individual amino acids contributions indicated by this panel. Φ-P-Φ, hydrophobic-proline-hydrophobic; APV, alanine-proline-valine; DYP, aspartic acid-tyrosine-proline; EEE, glutamic acid–glutamic acid–glutamic acid; EEI, glutamic acid–glutamic acid–isoleucine; EET, glutamic acid–glutamic acid–threonine; EGT, glutamic acid-glycine-threonine; EPA, glutamic acid-proline-alanine; EPT, glutamic acid-proline-threonine; EPV, glutamic acid-proline-valine; ER-ESCAPE motif, ER-Exit by Soluble Cargo using Amino-terminal Peptide-Encoding motif; EST, glutamic acid-serine-threonine; FGT, phenylalanine-glycine-threonine; FLT, phenylalanine-leucine-threonine; FPE, phenylalanine-proline-glutamic acid; FPR, phenylalanine-proline-arginine; FPT, phenylalanine-proline-threonine; FPV, phenylalanine-proline-valine; FQV, phenylalanine-glutamine-valine; FSM, phenylalanine-serine-methionine; FST, phenylalanine-threonine-valine; FTV, phenylalanine-threonine-valine; FVN, phenylalanine-valine-asparagine; GH, growth hormone; GPV, glycine-proline-valine; HSV, histidine-serine-valine; IEV, isoleucine-glycine-valine; IGV, isoleucine-glycine-valine; ILV, isoleucine-leucine-valine; INV, isoleucine-asparagine-valine; IPA, isoleucine-proline-alanine; IPD, isoleucine-proline-aspartic acid; IPE, isoleucine-proline-glutamic acid; IPP, isoleucine-proline-proline; IPS, isoleucine-proline-serine; IPV, isoleucine-proline-valine; IRV, isoleucine-arginine-valine; ISH, isoleucine-serine-histidine; ISP, isoleucine-serine-proline; ISQ, isoleucine-serine-glutamine; ISR, isoleucine-serine-arginine; ISV, isoleucine-serine-valine; ITV, isoleucine-threonine-valine; KAV, lysine-alanine-valine; KGV, lysine-glycine-valine; KSV, lysine-serine-valine; KVH, lysine-valine-histidine; NPV, asparagine-proline-valine; QPV, glutamine-proline-valine; QQV, glutamine-glutamine-valine; QSV, glutamine-serine-valine; RGV, arginine-glycine-valine; RLV, arginine-leucine-valine; RPK, arginine-proline-lysine; RPV, arginine-proline-valine; RRR, arginine-arginine-arginine; RSV, arginine-serine-valine; SLT, serine-leucine-threonine; SPT, serine-proline-threonine; SPV, serine-proline-valine; SRT, serine-arginine-threonine; SST, serine-serine-threonine; Surf4, surfeit locus protein 4; YPY, tyrosine-proline-tyrosine.
Fig 9.
When not in functional excess, Surf4 prioritizes ER exit of cargo with stronger ER-ESCAPE motif.
(A) Western blot analyses of cell extracts show steady-state levels of GH starting with strong ER-ESCAPE motif: APV < modest: ITV << nonbinding: EET. Addition of N-linked oligosaccharide (NTT) to fourth amino acid increased the mass/Mr but did not affect relative cellular steady-state levels for any of these proteins, (APV = APV-NTT) < (ITV = ITV-NTT) << (EET = EET-NTT). Coexpression of APV-GH and ITV-GH, irrespective of which construct was glycosylated, did not affect their respective steady-state levels. (B) GH detected in conditioned media (18 hr posttransfection) showed both the remaining complement of APV-GH was trafficked out of cells and the robust nature of HEK293A’s bulk flow process of nonbinding cargo (EET-GH). (C) Partial knockdown of Surf4 with siRNA depleted Surf4 protein levels sufficiently to show GH with strong binding ER-ESCAPE motif (APV-GH) outcompeting modestly binding GH (ITV-GH). HEK293A cells were pretreated for 36 hr with Surf4 siRNA before second transfection with noted GH constructs. Eighteen hr later, washed cells were processed for GH and Surf4 western blots. (D) Quantification of GH western blots shows modestly binding ITV-GH was less effectively trafficked than APV-GH when Surf4 levels were substantially depleted by siRNA. (Error bars are +/− SEM with sample size of n = 4 for inactive siRNA and n = 7 for Surf4 siRNA, P < 0.001.) Three μg cell lysate protein and 6% concentrated media were used for western blot analysis with goat anti-human GH. Six μg cell lysate protein was used for western blot analysis with rabbit anti-Surf4 carboxy-terminal peptide. LI-COR IR-fluorescent second antibodies were used for detection on LI-COR’s Odyssey scanner. Numbers on left are molecular weight standards in kDa. APV, alanine-proline-valine; EET, glutamic acid–glutamic acid–threonine; ER, endoplasmic reticulum; ER-ESCAPE motif, ER-Exit by Soluble Cargo using Amino-terminal Peptide-Encoding motif; GH, growth hormone; HEK293A, human embryonic kidney cell line 293; IR, infrared; ITV, isoleucine-threonine-valine; NTT, asparagine-threonine-threonine; siRNA, small interfering RNA; Surf4, surfeit locus protein 4.
Fig 10.
APV-GH interacts with digitonin-permeabilized Surf4 microsomes with half-maximal binding of 200–300 nM.
(A) Microsomes made from Surf4KO cells with (Lanes 1–4) or without (Lanes 5–8) expression of Surf4 expression by transfection for 24 hr. Half of the microsome aliquots (Lanes 3, 4, 7, and 8) were permeabilized with CEB (digitonin) for 30 min. As indicted, aliquots were incubated for 1 hr with 400 nM APV-GH or 400 nM EET-GH and briefly washed. Only combination of Surf4-expressing microsomes + digitonin + APV-GH resulted in significant increases (>5-fold) in detection by GH ELISA associated with the final >100,000 x g microsome pellet. (B) Preparation of microsomes from HA-Surf4-transfected HEK293A cells were incubated with magnetic beads precoated with antibodies to the cytosolic, carboxy-terminal domain of Surf4. Equal aliquots of Surf4 microsome beads were titrated with indicated concentration of digitonin (or CEB) for 30 min before incubation for 1 hr with 400 nM APV-GH, brief wash, and processing for GH ELISA analyses. Dose-response results show that CEB and ≥ 30 μg/ml digitonin were effective at permeabilizing microsomes for binding of APV-GH. Insert: Western blot shows that bead-associated microsomes contained both HA-Surf4 and the ERES marker, Sec23. (C) Equal aliquots of CEB-treated, Surf4 microsome/beads were incubated with increasing concentrations of APV-GH or EET-GH. APV-GH showed saturable binding characteristics with half-maximal binding at around 200–300 nM. EET-GH showed background levels of binding. (D) Equal aliquots of Surf4 microsome/beads were permeabilized with CEB (except first lane), incubated for 1 hr with 400 nM GH starting with indicated tripeptides, briefly washed, and analyzed by GH ELISA. Highest level of binding was with strong ER-ESCAPE motif APV-GH, followed by three modest binding motifs, FSM-GH, ISV-GH, and ITV-GH. The two acidic motifs, EET-GH and EEE-GH, bound at low levels also observed for microsome/beads not permeabilized by detergent. Each histogram bar represents mean ± SEM of transfections with each construct (n ≥ 7) with statistical comparisons to APV-GH (**p≤ 0.001, *p≤0.01) or to EET-GH (°°p ≤ 0.001). APV, alanine-proline-valine; CEB, Cytosol Extraction Buffer; EEE, glutamic acid–glutamic acid–glutamic acid; EET, glutamic acid–glutamic acid–threonine; ERES, ER exit site; ER-ESCAPE motif, ER-Exit by Soluble Cargo using Amino-terminal Peptide-Encoding motif; FSM, phenylalanine-serine-methionine; GH, growth hormone; HA, hemagglutinin; HEK293A, human embryonic kidney cell line 293; ISV, isoleucine-serine-valine; ITV, isoleucine-threonine-valine; Surf4, surfeit locus protein 4.
Fig 11.
Model illustrating interaction of ER-ESCAPE motifs with high, modest, and no affinity for Surf4/Erv29p.
(A) A green ball amino acid in spherical pocket denotes highest contribution of that residue to binding affinity such as a proline in number 2 position or a hydrophobic residue in position 1 (amino-terminus) or 3. Green half-ball plus red pyramid represents lower affinity interaction for that amino acid (e.g., serine in position 2), while the red cube denotes a negative contribution to binding affinity (e.g., acidic amino acid in position 1). High-affinity cargo present high-affinity contributions in all three positions, while modest- to low-affinity tripeptides have at least one mismatch. Nonbinding proteins such as chaperones or fibrillar collagens have two or three completely mismatching amino acids. (B) (1) High-affinity cargo (e.g., IPV) are bound to Surf4/Erv29p and exit ER before they accumulate to aggregate-forming concentrations. (2) Cargo with more modest ER-ESCAPE motifs (e.g., FSM) do not significantly bind to cargo receptor until (3) they accumulate to levels ≥ their binding constant. Only at that point do they remain bound long enough to remain in COPII vesicle at levels significantly greater than bulk flow. (4) illustrates cargo starting with nonbinding amino-terminal tripeptides (e.g., QEE) cannot exit ER more efficiently than their concentration in ER lumenal fluid in equilibrium with the small amount of exit vesicle fluid (bulk flow). (5) Fibrillar collagens are too large for standard COPII exit vesicles and must use more voluminous TANGO1/cTAGE5-associated exit vesicles. Large fibrillar collagens often start with nonbinding motif (e.g., QEE) to keep them from binding Surf4 and partially entering smaller COPII vesicles. COPII, coat protein complex II; ER, endoplasmic reticulum; ER-ESCAPE motif, ER-Exit by Soluble Cargo using Amino-terminal Peptide-Encoding motif; FSM, phenylalanine-serine-methionine; IPV, isoleucine-proline-valine; QEE, glutamine–glutamic acid–glutamic acid; Surf4, surfeit locus protein 1; TANGO1, transport and Golgi organization 1.
Fig 12.
Exit of highest affinity cargo is prioritized in ERES lacking an excess of Surf4/Erv29p.
Cargo receptors must have a high-affinity conformation to bind cargo in the ER and a low-affinity conformation to release cargo in fully formed exit vesicle or upon fusing with ERGIC/Golgi. High-affinity panel (A) illustrates a model whereby cargo receptors in the vicinity of ERESs have the ability to bind cargo before physically entering COPII vesicle, while the low-affinity panel (B) represents an alternative model in which the receptor is in its low-affinity state until interacting with elements of COPII vesicle (e.g., Sec24). In both cases, when there is an excess of cargo for local population of receptors, high-affinity cargo occupies available receptors, while lower-affinity cargo continues to build in concentration. This aids in keeping the most problematical proteins below their aggregation concentrations. Similarly, modest-affinity cargo, when their concentration becomes ≥ binding constant, occupy any available receptors before cargo with still-lower-affinity ER-ESCAPE motifs. This process delays aggregate formation until cargo receptors can be brought into balance with local/total cargo loading. Nonbinding cargo continue to exit solely by diffusion/equilibrium between fluids of COPII vesicle and ERES lumen (bulk flow). COPII, coat protein complex II; ER, endoplasmic reticulum; ERES, ER exit site; ER-ESCAPE motif, ER-Exit by Soluble Cargo using Amino-terminal Peptide-Encoding motif; ERGIC, ER-Golgi intermediate compartment; Erv29p, ER-derived vesicles protein 29; Surf4, surfeit locus protein 4.