ER LUMEN POSITIVE CHARGED PLUS

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. 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). The two acidic motifs, EET-GH and EEE-GH, bound at low levels also observed for microsome/beads not permeabilized by detergent. 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. (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. EET-GH showed background levels of binding. APV-GH showed saturable binding characteristics with half-maximal binding at around 200–300 nM. (C) Equal aliquots of CEB-treated, Surf4 microsome/beads were incubated with increasing concentrations of APV-GH or EET-GH. Insert: Western blot shows that bead-associated microsomes contained both HA-Surf4 and the ERES marker, Sec23. Dose-response results show that CEB and ≥ 30 μg/ml digitonin were effective at permeabilizing microsomes for binding of APV-GH. 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. (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. Error bars are SEM with sample size of n = 6 and P 5-fold) in detection by GH ELISA associated with the final >100,000 x g microsome pellet. The Luciferase activity was normalized to total protein (Luciferase units/mg protein).

Cells were harvested 22 hr posttransfection. Same proteins expressed in Surf4 KO cells retained their high steady-state levels. (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). Trafficking of di-acidic EET-GH was not rescued by HA-Surf4.

ER LUMEN POSITIVE CHARGED PLUS

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 Surf4 KO cells upon coexpression of HA-Surf4 protein. (H) Surf4-trafficked cargo with motifs other than Φ-P-Φ. In the bottom panel, AMELX (Myc-tagged) formed stable aggregate in Surf4 KO cells with most remaining in >100,000 x g pellet after solubilizing cells with an MEB for 10 min. As observed on western blots, 10 mM Ca 2+ stabilized a portion of DSPP in the pellet fraction.

Er lumen positive charged