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Role of cytoplasmic C-terminal amino acids of membrane proteins in ER export

¹ Biozentrum, University of Basel, CH-4056 Basel, Switzerland
² Department of Morphology, University of Geneva School of Medicine, CH-1211 Geneva, Switzerland
³ Tokyo University of Pharmacy and Life Science, 192-0392 Tokyo, Japan

View larger version (12K): [in a new window]   Fig. 6. Characterization of the valine transport signal. (A) Position dependence of valine. Tails of the indicated sequence were fused to the C-terminus of L53L18R2 (see Fig. 4). (B) The effect of neighboring amino acids on C-terminal valine-mediated transport of L53L18R2S8XX is shown. Acquisition of endo H resistance after pulse-chase was determined as in Fig. 1. Mean values±s.e.m. of at least four independent experiments are shown. An asterisk indicates statistical significance to the SV construct (P<0.05, Student's t-test).

View larger version (33K): [in a new window]   Fig. 1. The FF transport motif of ERGIC-53 can be functionally substituted by other motifs. (A) C-terminal amino acid sequences of ERGIC-53 from different species. Positions relative to the C-terminus are indicated. (B) A schematic representation of ERGIC-53 constructs expressed in COS cells. All constructs have an N-glycosylation site (CHO) at position 61 and a c-myc epitope (Itin et al., 1995). The TMD is followed by the amino-acid sequence of the cytoplasmic tail, in which the two lysines at position -3 and -4 were replaced by alanines to prevent recycling. The -1 and -2 positions (XX) were mutated to the amino acids indicated in panels C to E. (C) Effect of double substitutions of the FF motif. COS cells were transfected with the indicated constructs and subjected to pulse-chase/endo H analysis using [35S]-methionine. 60 minutes after the chase, the cells were lysed and ERGIC-53 constructs were immunoprecipitated with anti-myc. Immunoprecipitates were treated with endo H, separated by SDS-PAGE and analyzed by fluorography. The upper band represents the endo H-resistant and the lower band the endo H-sensitive form ERGIC-53. (D) Quantification of fluorograms shown in panel C. (E) Acquisition of endo H resistance of ERGIC-53 constructs with single motifs at the XX position. White bars in D an E represent values for the signalless reporter. Black bars represent values obtained with the FF construct. Mean values±s.d. of five independent experiments are shown.

View larger version (49K): [in a new window]   Fig. 2. Transport motifs bind to COPII components in vitro. Peptides comprising the sequence of ERGIC-53 tail with the indicated C-terminal motifs were coupled to thiol-activated Sepharose. The beads were incubated with detergent lysates of HepG2 cells. Bound proteins were separated by SDS-PAGE, and nitrocellulose blots were probed for COPII binding using antibodies against COPII subunits. (A) A binding assay with peptides bearing double substitutions at XX position. (B) A binding assay with peptides bearing single motifs. Lane C: lysate corresponding to one quarter of the amount incubated with peptide beads. Representative examples of at least three independent experiments are shown.

View larger version (18K): [in a new window]   Fig. 3. A C-terminal valine acts as a transport signal. (A) A schematic representation of CD4 reporter constructs. CD4 represents the luminal domain possessing two N-glycosylation sites; L18 represents 18 leucine TMD and X indicates mutated residues. (B) Effect of motifs at XX positions. COS cells were transfected with the indicated constructs, pulse-labeled with [35S]-methionine, chased for 60 minutes and subjected to immunoprecipitation with anti-CD4. Immunoprecipitates were digested with endo H (+) or left untreated (-), separated by SDS-PAGE and visualized by fluorography. The upper band in the endo H+ lanes is the endo-H-resistant and the lower band to the endo-H-sensitive form of the CD4 reporter. (C) Quantification of fluorograms. Means±s.e.m. of at least three independent experiments. Asterisk, difference to the AA values is statistically significant (student's t-test, P<0.05).

View larger version (20K): [in a new window]   Fig. 4. Transport of ERGIC-53-based reporters. The luminal domain (L53) derived from ERGIC-53 carries a c-myc tag and an N-glycosylation site. L53 constructs with an ERGIC-53 wild-type TMD are designated L53T53 and constructs with an 18 leucine TMD L53L18. (A) Substitution of the TMD and cytoplasmic domain of ERGIC-53 does not interfere with oligomerization. 48 hours after transfection, the cells were labeled for 5 minutes with [35S]methionine and chased for 15 minutes. Cells were washed and lysed in the presence of 20 mM iodoacetamide before immunoprecipitation with anti-myc. Immunoprecipitates were separated by 4-10% gradient SDS-PAGE under reducing (+DTT) or non-reducing (-DTT) conditions followed by fluorography. M, Mr marker lane. (B) Transport efficiency of constructs probed by pulse-chase/endo H (Fig. 1). This analysis also included monomeric forms of constructs L53L18R2S10 and L53L18R2S9V denoted mL53L18R2S10 and mL53L18R2S9V. The white and black bars represent the same controls as in Fig. 1. Means±s.e.m. of at least three independent experiments.

View larger version (135K): [in a new window]   Fig. 5. A C-terminal valine mediates transport of a signalless reporter to the cell surface. The localization of oligomeric and monomeric ERGIC-53 constructs with and without a valine transport signal was analyzed by immunofluorescence microscopy. COS cells were transfected with oligomeric (A to F) or monomeric (G to L) constructs L53L18R2S10 (D to F, J to L) or L53L18R2S9V (A to C, G to I) and processed for immunofluorescence microscopy 20 hours later. Panels A, D, G and J (bar, 50 µm): surface labeling of nonpermeabilized cells. Other panels: double staining after permeabilization (bar, 25 µm). Panels A, B, D, E, G, H, J and K: staining with anti-myc. Panels C, F, I and L: staining with a mAb against the ER marker BAP31.

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