Cargo selection into COPII vesicles is driven by the Sec24p subunit

Abstract

Transport of secretory proteins out of the endoplasmic reticulum (ER) is mediated by vesicles generated by the COPII coat complex. In order to understand how cargo molecules are selected by this cytoplasmic coat, we investigated the functional role of the Sec24p homolog, Lst1p. We show that Lst1p can function as a COPII subunit independently of Sec24p on native ER membranes and on synthetic liposomes. However, vesicles generated with Lst1p in the absence of Sec24p are deficient in a distinct subset of cargo molecules, including the SNAREs, Bet1p, Bos1p and Sec22p. Consistent with the absence of any SNAREs, these vesicles are unable to fuse with Golgi membranes. Furthermore, unlike Sec24p, Lst1p fails to bind to Bet1p in vitro, indicating a direct correlation between cargo binding and recruitment into vesicles. Our data suggest that the principle role of Sec24p is to discriminate cargo molecules for incorporation into COPII vesicles.

Fig. 1. Sec23/Lst1p can form a COPII coat on synthetic liposomes. Liposomes (200 µM lipids) composed of either microsomal lipids or synthetic lipids were incubated with COPII components (20 µg/ml Sar1p, 30 µg/ml Sec23/24p or Sec23/Lst1p, 45 µg/ml Sec13/31p) and nucleotide (100 µM) as indicated. Liposomes were collected by flotation and bound proteins were analyzed by SDS–PAGE and SYPRO red staining. A portion of the total reactions was loaded in lanes T9 and T14. The protein marked by an asterisk is a proteolytic fragment of Sec31p.

Fig. 2. Real-time recruitment of the COPII coat. (A–C) The light scattering of a suspension of major–minor liposomes (100 µM lipids) was monitored continuously upon the addition of 930 nM Sar1p, 160 nM Sec23/24p or 160 nM Sec23/Lst1p, 260 nM Sec13/31p and 100 µM GMP-PNP (A and B) or GTP (C) at specific time points as indicated. (D) GAP activity of the Sec23/24p or Sec23/Lst1p complex (55 nM) on Sar1p (2 µM) pre-incubated with 100 µM GTP and major–minor liposomes (300 µM lipids) was determined by tryptophan fluorescence in the absence and presence of Sec13/31p (60 nM).

Fig. 3. Sec23/Lst1p supports budding from synthetic liposomes. Liposomes (100 µg) extruded through 400 nm filters were used in budding reactions in the presence of Sar1p (85 µg/ml), Sec23/24p (85 µg/ml) or Sec23/Lst1p (85 µg/ml), Sec13/31p (100 µg/ml) and GMP-PNP (100 µM). Budded vesicles (fractions 11 and 12) were separated from large, coated liposomes (fractions 3–7) on linear sucrose gradients, and the distribution of lipid quantified by NBD-phospholipid fluorescence.

Fig. 4. Sec23/Lst1p packages a subset of cargo proteins. (A) Total membranes (T) and budded vesicles were collected from budding reactions containing COPII subunits as indicated. Proteins were analyzed by SDS–PAGE and autoradiography (for ³⁵S-labeled α-factor) or immunoblotting with antibodies against a variety of cargo molecules. Lipid release was quantified by TLC and staining of lipids; numbers given are the percentage of total lipid released into the vesicle fraction. (B) Release of ³⁵S-labeled α-factor into a vesicle fraction after incubation of membranes with Sar1p, Sec23/24p or Sec23/Lst1p, Sec13/31p and guanine nucleotide was determined by precipitation of glycosylated α-factor from the medium speed supernatant. (C) Vesicles generated as in (B) in the presence of either Sec23/24p or Sec23/Lst1p and GTP were incubated with acceptor Golgi membranes in the absence or presence of cytosol. Fusion of vesicles with the Golgi was determined by precipitation with antibodies against α-1,6 mannose residues and scintillation counting.

Fig. 5. Sec23/Lst1p vesicles are morphologically identical to Sec23/24p vesicles. Thin-section microscopy of the vesicle-enriched fractions from budding reactions performed with either Sec23/24p (A) or Sec23/Lst1p (B). The overall vesicle morphology and coat appearance are comparable in both samples. The mean size of vesicles generated with Sec23/24p (72.1 ± 14 nm) was moderately smaller than that of those generated with Sec23/Lst1p (75.4 ± 16 nm; P < 0.002). Scale bar = 100 nm.

Fig. 6. Recruitment of proteins into pre-budding complexes mirrors vesicle packaging. (A and B) Cargo-enriched pre-budding complexes were isolated from urea-washed membranes after incubation with GST–Sar1p and COPII subunits as indicated. A proportion (1%) of total membranes is included for comparison. The precursor (p) and Golgi-modified mature (m) forms of Gas1p are indicated.

Fig. 7. Sec23/Lst1p does not bind Bet1p. (A) The cytoplasmic domain of the v-SNARE, Bet1p, fused to GST was incubated with Sar1p and either Sec23/24p or Sec23/Lst1p and guanine nucleotide as indicated. Bound proteins were analyzed by SDS–PAGE and SYPRO red staining. (B) Full-length Bet1p was incorporated into neutral liposomes and binding of Sec23/24p or Sec23/Lst1p was assessed by flotation. For comparison, neutral liposomes were either hydrated in buffer and extruded immediately (PC/PE) or hydrated in detergent solution in the absence of Bet1p, dialyzed and extruded as for those containing Bet1p (mock). COPII components were incubated with liposomes as indicated, and proteins bound to liposomes were detected by SDS–PAGE and SYPRO red staining. A portion of the total reactions was loaded in lanes T1 and T3. The protein marked by an asterisk is a proteolytic fragment of Sec31p.