Role of Sec24 isoforms in selective export of membrane proteins from the endoplasmic reticulum

Abstract

Sec24 of the COPII (coat protein complex II) vesicle coat mediates the selective export of membrane proteins from the endoplasmic reticulum (ER) in yeast. Human cells express four Sec24 isoforms, but their role is unknown. Here, we report the differential effects of Sec24 isoform-specific silencing on the transport of the membrane reporter protein ERGIC-53 (ER-Golgi intermediate compartment-53) carrying the cytosolic ER export signals di-phenylalanine, di-tyrosine, di-leucine, di-isoleucine, di-valine or terminal valine. Knockdown of single Sec24 isoforms showed dependence of di-leucine-mediated transport on Sec24A, but transport mediated by the other signals was not affected. By contrast, double knockdown of Sec24A with one of the other three Sec24 isoforms impaired all aromatic/hydrophobic signal-dependent transport. Double knockdown of Sec24B/C or Sec24B/D preferentially affected di-leucine-mediated transport, whereas knockdown of Sec24C/D affected di-isoleucine- and valine-mediated transport. The isoform-selective transport correlated with binding preferences of the signals for the corresponding isoforms in vitro. Thus, human Sec24 isoforms expand the repertoire of cargo for signal-mediated ER export, but are in part functionally redundant.

Figure 1

Effects of Sec24 isoform-specific knockdown on signal-mediated transport. (A) A Myc-tagged glycosylated variant of human ERGIC-53 was used as a reporter for endoplasmic reticulum to Golgi transport. The −1 and −2 positions (XX) in the cytoplasmic tail were replaced by the amino acids indicated in (C) and (D). CHO, N-glycosylation site; TMD, transmembrane domain. (B) HeLa cells were transfected with equal amounts of siRNAs specific for the individual Sec24 isoforms or control (Ctr) siRNA. After 3 days, the cells were subjected to SDS–polyacrylamide gel electrophoresis (SDS–PAGE). Immunoblots were probed for the four Sec24 isoforms (Sec24A, Sec24B, Sec24C, Sec24D), Sec23 and tubulin as a loading control. (C) One day after siRNA transfection, the cells were transfected with the ERGIC-53 reporter carrying the indicated amino-acid doublets at the XX position shown in (A); AA, FF, YY, LL, II, VV and AV. After another 2 days, the cells were pulsed for 10 min with [³⁵S]methionine, chased for 1 h and then subjected to immunoprecipitation with Myc antibodies. Precipitates were treated with endo H and separated by SDS–PAGE. Endo H-resistant (arrow) and endo H-sensitive (arrowhead) forms of the ERGIC-53 constructs were visualized by using fluorography, and the relative amount of endo H-resistant form was quantified (D). Values are means±s.d.; n=3–5; statistically significant difference to control siRNA (P<0.05; Student's t-test). AA, di-alanine; AV, alanine–valine; ERGIC, endoplasmic reticulum–Golgi intermediate compartment; FF, di-phenylalanine; II, di-isoleucine; LL, di-leucine; siRNA, short interfering RNA; VV, di-valine; YY, di-tyrosine.

Figure 2

Effects of double knockdown of Sec24 isoforms on signal-mediated transport. (A) HeLa cells were transfected with specific siRNAs against two of the four Sec24 isoforms or with control (Ctr) siRNA. After 3 days, the cells were analysed by immunoblotting, as in Fig 1. (B–H) Quantification of the maturation of ERGIC-53 constructs carrying the indicated transport signals after knockdown of Sec24 isoform pairs. Values are means±s.d.; n=3–5; statistically significant difference to control siRNA (P<0.05; Student's t-test). AA, di-alanine; AV, alanine–valine; ERGIC, endoplasmic reticulum–Golgi intermediate compartment; FF, di-phenylalanine; II, di-isoleucine; LL, di-leucine; siRNA, short interfering RNA; VV, di-valine; YY, di-tyrosine.

Figure 3

Effects of triple and quadruple knockdown of Sec24 isoforms on signal-mediated transport. (A) HeLa cells were transfected with siRNAs against combinations of three or all four Sec24 isoforms and analysed by immunoblotting. (B–H) The maturation of the indicated ERGIC-53 constructs was quantified after triple Sec24 isoform knockdown. (I) Knockdown of all four Sec24 isoforms. A representative fluorogram (left panel) and the quantification of 3–5 fluorograms are shown (right panel). Triple and quadruple Sec24 knockdown combinations result in a significant decrease of endoplasmic reticulum export for all constructs when compared with control (Ctr) siRNA experiments (n=3–5; values are means±s.d.; P<0.05; Student's t-test). AA, di-alanine; AV, alanine–valine; ERGIC, endoplasmic reticulum–Golgi intermediate compartment; FF, di-phenylalanine; II, di-isoleucine; LL, di-leucine; siRNA, short interfering RNA; VV, di-valine; YY, di-tyrosine.

Figure 4

Sec24D overexpression does not compensate for the loss of Sec24A and Sec24B. (A,B) HeLa cells were transfected with siRNA against Sec24A (A) or Sec24A and Sec24B (B). After 1 day, the cells were co-transfected with Myc–Sec24D and Myc–ERGIC-53 constructs carrying LL (A) or FF (B). After another 2 days, the transport assay was performed (see Fig 1C) and endo H-resistant (arrow) and endo H-sensitive (arrowhead) forms of the ERGIC-53 constructs were visualized by fluorography. Immunoprecipitation of the endogenous marker BAP31 was used as a loading control (Ctr). (C,D) Quantification of fluorograms shown in (A) and (B). Values are means±s.d.; n=3. AA, di-alanine; AV, alanine–valine; ERGIC, endoplasmic reticulum–Golgi intermediate compartment; FF, di-phenylalanine; II, di-isoleucine; LL, di-leucine; siRNA, short interfering RNA; VV, di-valine; YY, di-tyrosine.

Figure 5

Binding of Sec24 isoforms to transport signals in vitro. ERGIC-53 tail peptides carrying the indicated motifs were coupled to thiol-activated Sepharose and the beads were incubated with precleared HeLa cell lysate. Bound proteins were separated by SDS–polyacrylamide gel electrophoresis and immunoblotted for the four Sec24 isoforms (Sec24A–Sec24D), Sec23 and tubulin. A representative immunoblot of four independent experiments is shown. (B) Quantification of immunoblots shown in (A). Signal intensities for the Sec24 isoforms were related to those of the respective input signal (A). Values are means±s.d.; n=4. (C) The binding preferences of each Sec24 isoform for the different motifs are depicted from high preference (+++) to lowest preference (−). Lowest preference means similar to AA. AA, di-alanine; AV, alanine–valine; ERGIC, endoplasmic reticulum–Golgi intermediate compartment; FF, di-phenylalanine; II, di-isoleucine; LL, di-leucine; VV, di-valine; YY, di-tyrosine.