The Erv41p–Erv46p complex: multiple export signals are required in trans for COPII-dependent transport from the ER

Abstract

Erv41p and Erv46p form an integral membrane protein complex that cycles between the endoplasmic reticulum (ER) and Golgi. Both proteins contain a large lumenal domain and short N-and C-terminal tail sequences exposed to the cytosol. The coat protein complex II (COPII) packages the Erv41p±Erv46p complex into ER-derived vesicles for delivery to the Golgi. We determined signals in the Erv41p±Erv46p complex that are required for COPII-dependent export from the ER. Mutants lacking the Erv41p or Erv46p C-terminus accumulated in the ER and were not packaged ef®ciently into vesicles. We identi®ed an isoleucine±leucine sequence in the Erv41p tail that was required for COPII binding and inclusion of the complex into vesicles. This signal was suf®cient for COPII binding but not for ER export. The Erv46p tail contains a phenylalanine±tyrosine sequence required together with the isoleucine±leucine signal in Erv41p for export of the complex. Surprisingly, Erv41p± Erv46p tail-swapped chimeras were not exported from the ER, indicating that signals in both the Erv41p and the Erv46p tail sequences are required in a speci®c orientation for ef®cient packaging of the Erv41p±Erv46p complex.

Molecular Dissection of Erv26p Identifies Separable Cargo Binding and Coat Protein Sorting Activities

“…These observations indicate that the C-terminal HA tag was also sensitive to proteinase K and therefore was cytosolically exposed, consistent with previous evidence for antibody accessibility to the C terminus of Erv26p (14). As controls for membrane integrity and proteinase K activity in these experiments, Erv46p, a transmembrane protein with relatively short cytosolic segments and a large protected lumenal domain (27), as well as the cytosol-facing SNARE protein Bos1p (28) were monitored. Upon protease treatment, Erv46p shifted to a protease-protected species of the expected size (27), whereas Bos1p was fully digested (lanes 3 and 7).…”

Mammalian Erv46 localizes to the endoplasmic reticulum–Golgi intermediate compartment and to cis-Golgi cisternae

“…Erv46p contains a canonical COPI binding motif for retrograde transport, a feature that is conserved among all of its homologs in other species. Anterograde transport by COPII requires a hydrophobic COPII binding motif on Erv41p in concert with a diaromatic motif on Erv46p (24). Therefore, we propose that the C-terminal tail sequences of a mammalian mErv41-mErv46 complex are responsible for its observed localization through dynamic associations with COPI and COPII.…”

“…These proteins share a conserved membrane topology, a dilysine COPI binding motif (22,23), and eight completely invariant cysteine residues. Most of Erv46p is luminally oriented with two membrane spanning segments and short N and C termini that are cytoplasmically exposed and are required for COPII binding and sorting into COPII vesicles (24). Overall, mErv46 and yeast Erv46p sequences consist of 37% identical and 52% similar amino acid residues.…”

Proteomics of Endoplasmic Reticulum-Golgi Intermediate Compartment (ERGIC) Membranes from Brefeldin A-treated HepG2 Cells Identifies ERGIC-32, a New Cycling Protein That Interacts with Human Erv46

“…Like ERGIC-32, hErv41 and hErv46 lack a cleavable signal sequence and are predicted to have large luminal and short NH 2 -and COOH-terminal cytosolic domains. Transport signals present in the COOH-terminal part of Erv41p and Erv46p proteins and acting in trans are responsible for the COPII-dependent exit of the complex from the ER and its cycling between the ER and Golgi in yeast (75). Two hydrophobic residues, Ile-349 and Leu-350 in positions Ϫ4 and Ϫ3 of the COOH terminus of Erv41p, constitute a transport motif required for the recruitment of the Erv41p-Erv46p complex into COPII vesicles.…”

“…A di-leucine motif is present in positions Ϫ6 and Ϫ7 of mammalian hErv41 and an Ile-Tyr sequence is located at the same distance from the membrane in hErv46 as the Phe-Tyr sequence in yeast Erv46p. A di-lysine motif in positions Ϫ3 and Ϫ4 of the Erv46p may mediate the Golgi to ER retrograde trafficking of the Erv41p-Erv46p complex (66,75). Only the lysine in position Ϫ3 is conserved in mammalian Erv46.…”