Concentrative sorting of secretory cargo proteins into COPII-coated vesicles

Abstract

Here, we show that efficient transport of membrane and secretory proteins from the ER of Saccharomyces cerevisiae requires concentrative and signal-mediated sorting. Three independent markers of bulk flow transport out of the ER indicate that in the absence of an ER export signal, molecules are inefficiently captured into coat protein complex II (COPII)-coated vesicles. A soluble secretory protein, glycosylated pro-alpha-factor (gpalphaf), was enriched approximately 20 fold in these vesicles relative to bulk flow markers. In the absence of Erv29p, a membrane protein that facilitates gpalphaf transport (Belden and Barlowe, 2001), gpalphaf is packaged into COPII vesicles as inefficiently as soluble bulk flow markers. We also found that a plasma membrane protein, the general amino acid permease (Gap1p), is enriched approximately threefold in COPII vesicles relative to membrane phospholipids. Mutation of a diacidic sequence present in the COOH-terminal cytosolic domain of Gap1p eliminated concentrative sorting of this protein.

Figure 1.

A soluble secretory protein (gpαf) is enriched in COPII vesicles relative to markers of bulk flow transport. (A) Microsomal membranes prepared from cells expressing GFP-HDEL (RSY255/pDN330) were combined with purified COPII proteins in the presence (+) or absence (−) of GMP-PNP (NTP). Serial dilutions of the total reaction (Total) and a vesicle-enriched (Vesicle) fraction were collected and analyzed by SDS-PAGE and immunoblotting using anti-GFP antibodies. (B) ³⁵S-prepro–α-factor was translocated into microsomal membranes and these membranes were used in in vitro COPII-budding assays as described in A. The amount of gpαf in the vesicle fraction was quantified by PhosphorImager analysis. (C) Iodinated acyltripeptides were translocated into microsomal membranes prepared from RSY255. These membranes were used in in vitro COPII-budding assays, and the amount of membrane-associated glycosylated tripeptide (g-peptide) packaged into vesicles was quantified by scintillation counting (see Materials and methods). (D) Total (5%) and Vesicle (75%) fractions were collected from budding reactions containing all five purified COPII proteins (+) or lacking Sar1p (−). Fractions were analyzed for gpαf content by SDS-PAGE and for phospholipid content by organic extraction, followed by TLC and staining with a fluorescent dye (see Materials and methods). The percentage of phosphatidylcholine (PC) and phosphatidylserine (PS) (averaged) packaged into COPII-coated vesicles is shown in E. (F) ³⁵S-prepro–α-factor was translocated into microsomal membranes prepared from ERV29 (BY4742) and erv29Δ (Y15936) cells. These membranes were used in in vitro COPII-budding assays as described in D and quantified as in B.

Figure 2.

A plasma membrane precursor protein requires an ER export signal for enrichment into COPII vesicles. (A) Microsomal membranes prepared from cells grown in SUD expressing HA-tagged Gap1p (Y17050/pPM11) or Gap1(X564–7A)p (Y17050/pPM12) were used in in vitro COPII-budding assays as described in the legend to Fig. 1 D. 5% of the total reaction (T), and 75% of the vesicle fraction (V) were analyzed by SDS-PAGE and immunoblotting (top two panels). A ³⁵S-labeled secondary antibody and PhosphorImager analysis were used to quantify the packaging of Sec22p and Gap1p into COPII-coated vesicles (B). Phospholipids (A, bottom) were analyzed as in Fig. 1 D, and the percentage phospholipids packaged into COPII-coated vesicles was determined (B, right).

Figure 3.

Characterization of a diacidic ER export signal in the COOH-terminal domain of Gap1p. (A) The COOH- terminal domain of Gap1p (amino acids 546–602) was used in a BLAST search of the S. cerevisiae proteome to find related sequences (see Materials and methods). Gap1p and the 13 amino acid permeases identified in the BLAST search were aligned using ClustalW. Residues shaded black are identical in >80% of the aligned sequences and those shaded gray are similar in >80% of the aligned sequences. (B) Cells expressing HA-tagged Gap1p constructs were metabolically labeled for 3 min. Semi-intact cells (SICs) prepared from labeled cells were used in in vitro COPII-budding assays and analyzed by immunoprecipitation, SDS-PAGE, and PhosphorImager analysis. The packaging of Gap1p diacidic mutant proteins was normalized to the packaging of wild-type Gap1p.

Figure 4.

Packaging of Gap1/Can1p chimeras into COPII vesicles. The first 535 residues of Gap1p and 540 residues of Can1p (NH₂-terminal region) were fused to the cytosolic COOH-terminal domain of Gap1p, Gap1(D564A)p, or to a truncation possessing the first seven residues of the Gap1p COOH-terminal domain followed by two serines and a stop codon (ΔCT). HA-tagged chimeras were expressed from multicopy plasmids in BY4742 cells. Radiolabeled SICs prepared from these cells were used in in vitro COPII-budding assays (as described in the legend to Fig. 3 B). The percentage of radiolabeled chimera packaged into COPII vesicles is plotted on the right.