Characterization of aromatic residue-controlled protein retention in the endoplasmic reticulum of Saccharomyces cerevisiae

Abstract

An endoplasmic reticulum (ER) retention sequence (ERS) is a characteristic short sequence that mediates protein retention in the ER of eukaryotic cells. However, little is known about the detailed molecular mechanism involved in ERS-mediated protein ER retention. Using a new surface display-based fluorescence technique that effectively quantifies ERS-promoted protein ER retention within Saccharomyces cerevisiae cells, we performed comprehensive ERS analyses. We found that the length, type of amino acid residue, and additional residues at positions -5 and -6 of the C-terminal HDEL motif all determined the retention of ERS in the yeast ER. Moreover, the biochemical results guided by structure simulation revealed that aromatic residues (Phe-54, Trp-56, and other aromatic residues facing the ER lumen) in both the ERS (at positions -6 and -4) and its receptor, Erd2, jointly determined their interaction with each other. Our studies also revealed that this aromatic residue interaction might lead to the discriminative recognition of HDEL or KDEL as ERS in yeast or human cells, respectively. Our findings expand the understanding of ERS-mediated residence of proteins in the ER and may guide future research into protein folding, modification, and translocation affected by ER retention.

Keywords: ER retention sequence; Erd2; endoplasmic reticulum (ER); flow cytometry; fluorescence; protein ER retention; protein sorting; protein trafficking (Golgi); surface display; yeast.

Figure 1.

Scheme of the strategy for evaluating the ERS-promoted ER retention effect in yeast. Cells bearing Aga2–FLAG–ERS cassettes were labeled with iFluor 647–conjugated anti-FLAG antibody followed by flow cytometry and fluorescence microscopy analysis. The red arrows (top right) point toward the yeast cells with surface fluorescence. w/o, without ERS; w/, with ERS.

Figure 2.

Characterization of the ER retention strength of different FEHDEL derivatives. The SD efficiencies of the Aga2–FLAG–ERS cassettes containing different FEHDEL derivatives were quantitated using FACS analysis after induction at 20 °C (A and B) or in a time course of 24–48 h after induction at 30 °C (C) or 20 °C (D). E, the in-cell mRNA levels of the Aag2–FLAG cassettes without ERS or with C-terminal ERS of HDEL, FEHDEL, and WEHDEL were quantitated by RT-PCR after induction for 6 h at 30 °C. F, the normalized SD efficiency of the sequences of NFRDEL, DGEDEL, PYLDEL, and MLKDEL was evaluated after induction at 20 °C. The induced cells were surface-labeled with iFluor 647–conjugated anti-FLAG antibodies. Data are presented as mean ± S.E. (n = 3 independent experiments). In A, B, and F, the data were normalized with cells bearing the Aga2–FLAG cassette. p ≤ 0.05 (Student's t test).

Figure 3.

Interaction of different ERS with Erd1 and Erd2. A–D, under conditions with or without overexpressed Erd1 or Erd2, the SD efficiencies of the Aga2–GFP–FLAG (A) or Aga2–GFP–FLAG–ERS cassettes (B–D) (ERS: HDEL, FEHDEL, or WEHDEL) were recorded at 24 h after induction at 30 °C. E, the mRNA levels of ERD1 and ERD2 in the cells bearing Aga2–GFP–FLAG or Aga2–GFP–FLAG–WEHDEL cassettes were quantitated by RT-PCR after induction for 6 h at 30 °C. The cells were surface-labeled with iFluor 647–conjugated anti-FLAG antibodies for SD efficiency quantitation. The data are presented as mean ± S.E. (n = 3 independent experiments). p ≤ 0.05 (Student's t test).

Figure 4.

Exploring the position of ERS in a protein for its interaction with ERD2. A and B, the normalized SD efficiency (A) or total cellular GFP fluorescent intensity (B) of the different Aga2–GFP–FLAG–ERS and Aga2–ERS–GFP–FLAG cassettes was analyzed after induction for 6 h at 30 °C. C, the in-cell mRNA levels of the Aag2–GFP–FLAG cassettes without ERS or with C-terminal ERS of HDEL, FEHDEL, and WEHDEL were quantitated by RT-PCR after induction for 6 h at 30 °C. D, the proposed scheme presents the interaction between Erd2 and ERS located at the C terminus of a protein complex. E, the intracellular and surface-localized GFP fluorescence of cells bearing the Aga2–GFP–FLAG or different Aga2–GFP–FLAG–ERS cassettes. The red arrows point to the accumulation of GFP fluorescence in yeast ER and Golgi. The cells were surface-labeled with iFluor 647–conjugated anti-FLAG antibodies for SD efficiency quantitation. In A and B, data are presented as mean ± S.E. (n = 3 independent experiments). p ≤ 0.05 (Student's t test).

Figure 5.

Structural simulation of the interaction between Erd2 and ERS. A, the overall simulated structure of Erd2 using the I-TASSER program. B, the aromatic residues located on loop 1 (blue), loop 2 (purple), and loop 3 (red) form a characteristic subdomain facing the ER lumen. C–E, the speculated interaction between Erd2 and FEHDEL (C), WEHDEL (D), and FEKDEL (E). F, the FEHDEL polypeptide was embedded into the aromatic subdomain with the His residue inserted into the cavity of Erd2.

Figure 6.

Characterization of the key residues in the interaction between HDEL-type ERS and Erd2. A–D, under conditions without overexpression of ERD2 or with overexpression of ERD2 or its mutants, the SD efficiency of the Aga2–FLAG–ERS and Aga2–FLAG–ERS cassettes (ERS: HDEL, FEHDEL, or WEHDEL in supplemental Table S1) was quantitated after induction for 3 and 6 h at 30 °C, respectively. The cells were surface-labeled with iFluor 647–conjugated anti-FLAG antibodies for FACS analysis. The red or blue dotted line represents the SD efficiency of the Aga2–FLAG–WEHDEL cassette in a normal yeast cell or with overexpressed Erd2, for comparison, respectively. E, the hypothesized mechanism for the interaction between HDEL-type ERS and Erd2 in S. cerevisiae. In A–D, the data are presented as mean ± S.E. (n = 3 independent experiments). p ≤ 0.05 (Student's t test).

Figure 7.

Application of WEHDEL using the YESS approach. The MMP7 against its native substrate, RPLALWRS, and the human IgG lower hinge sequence, PAPELLGGP, were tested using the YESS approach with different ERS. A, without ERS; B, FEHDEL; C, WEHDEL. Induced cells were surface-labeled with iFluor 647–conjugated anti-FLAG antibodies and FITC-conjugated anti-HA antibodies followed by FACS analysis. High APC fluorescence with little or no FITC fluorescence indicated the specific cleavage at the substrate sequence (16).