Lst1p and Sec24p cooperate in sorting of the plasma membrane ATPase into COPII vesicles in Saccharomyces cerevisiae

Abstract

Formation of ER-derived protein transport vesicles requires three cytosolic components, a small GTPase, Sar1p, and two heterodimeric complexes, Sec23/24p and Sec13/31p, which comprise the COPII coat. We investigated the role of Lst1p, a Sec24p homologue, in cargo recruitment into COPII vesicles in Saccharomyces cerevisiae. A tagged version of Lst1p was purified and eluted as a heterodimer complexed with Sec23p comparable to the Sec23/24p heterodimer. We found that cytosol from an lst1-null strain supported the packaging of alpha-factor precursor into COPII vesicles but was deficient in the packaging of Pma1p, the essential plasma membrane ATPase. Supplementation of mutant cytosol with purified Sec23/Lst1p restored Pma1p packaging into the vesicles. When purified COPII components were used in the vesicle budding reaction, Pma1p packaging was optimal with a mixture of Sec23/24p and Sec23/Lst1p; Sec23/Lst1p did not replace Sec23/24p. Furthermore, Pma1p coimmunoprecipitated with Lst1p and Sec24p from vesicles. Vesicles formed with a mixture of Sec23/Lst1p and Sec23/24p were similar morphologically and in their buoyant density, but larger than normal COPII vesicles (87-nm vs. 75-nm diameter). Immunoelectronmicroscopic and biochemical studies revealed both Sec23/Lst1p and Sec23/24p on the membranes of the same vesicles. These results suggest that Lst1p and Sec24p cooperate in the packaging of Pma1p and support the view that biosynthetic precursors of plasma membrane proteins must be sorted into ER-derived transport vesicles. Sec24p homologues may comprise a more complex coat whose combinatorial subunit composition serves to expand the range of cargo to be packaged into COPII vesicles. By changing the geometry of COPII coat polymerization, Lst1p may allow the transport of bulky cargo molecules, polymers, or particles.

Figure 1

Purification of Sec23p complexes. Heterodimers of Sec23/Iss1p (lane I), Sec23/Lst1p (lane L) and Sec23/24p (lane 24) were purified as described in Materials and Methods and separated on a 7.5% SDS-PAGE gel followed by Sypro Red staining (A) or immunoblot analysis (B) with either anti-Iss1p, anti-Lst1p, or anti-Sec24p antibodies as indicated.

Figure 2

α-Factor and Pma1p packaging into vesicles reconstituted with cytosol. Budding assays were performed in duplicates as described in Materials and Methods using donor membranes from lst1Δ, HA-Pma1p-containing cells primed with wild-type cytosol, or lst1Δ cytosol supplemented with Sec23/Lst1p as indicated. Percent packaging of α-factor and Pma1p was determined as described in Materials and Methods and is reported as the mean of at least two independent experiments with error bars indicating SD.

Figure 3

α-Factor and Pma1p packaging into vesicles reconstituted with all purified COPII components. Budding was performed in duplicates using lst1Δ, HA-Pma1p containing SICs primed with GTP, 20 μg/ml Sar1p, and 40 μg/ml Sec13/31p, plus the indicated concentrations of Sec23/24p and Sec23/Lst1p. Percent packaging of α-factor and Pma1p was determined as described in Materials and Methods and is reported as the mean of at least two independent experiments with error bars indicating SD.

Figure 6

Conventional thin section samples from enriched fractions of COPII-coated vesicles produced from lst1-null microsomes in reactions containing Sec23/24p alone (20 μg/ml; A) or Sec23/Lst1p and Sec23/24p (10 μg/ml each; B). The overall vesicles morphology and the coat appearance are comparable in both incubations; the coated vesicles generated in mixture B are of slightly larger mean diameter (87 ± 14 nm SD) than those produced in mixture A (75 ± 10 nm SD). 800 vesicles were measured in each condition. The fractions also contain large vacuolar-type uncoated membrane contaminants. Bars, 100 nm.

Figure 4

Coimmunoprecipitation of HA-Pma1p with Lst1p and Sec24p. Vesicles were generated from lst1-null, HA-Pma1p containing SICs using either a mixture of 10 μg/ml each of Sec23/Lst1p and Sec23/24p (lanes 1) or 20 μg/ml Sec23/24p only (lanes 2). In lanes 3, a mixture of 10 μg/ml each of Sec23/Lst1p and Sec23/24p was used as in lanes 1, but wild-type SICs membranes, not expressing HA-tagged Pma1p, were used. Lanes 4 contained a mixture of the vesicles generated in reactions 2 and 3. After cross-linking, reactions were immunoprecipitated with anti-Lst1p or anti-Sec24p as indicated, and subjected to SDS-PAGE and immunoblot analysis using anti-HA antibodies. Input lanes represent 1% of the cross-linked reactions before immunoprecipitation.

Figure 5

Density gradient analysis of COPII vesicles. Four large-scale budding reactions, using lst1-null membranes, Sar1p, Sec13/31p, and the indicated combinations of Sec23/24p (Sec24p) and Sec23/Lst1p (Lst1p) at a concentration (combined) of 5 μg/ml, were performed (A–D). Vesicles were floated in sucrose gradients as described in Materials and Methods. Fractions were either subjected to SDS-PAGE followed by immunoblot analysis with the indicated antibodies and exposed simultaneously to film, or counted in a scintillation counter for [³⁵S]gpαF distribution.

Figure 8

Coimmunoprecipitation of Lst1p and Sec24p is membrane-dependent. Reactions containing purified COPII components and a mixture of both Sec23/Lst1p and Sec23/24p (lanes 1) or Sec23/24p only (lanes 2) were set in the presence (+memb) or absence (−memb) of donor membranes and supernatants containing vesicles (+memb) or only soluble COPII proteins (−memb) were cross-linked and centrifuged as described under Materials and Methods. Solublized pellets were then either immunoprecipitated with anti-Lst1p serum and immunoblotted with anti-Sec24p serum (A) or vice versa (B). Purified Sec23/24p and Sec23/Lst1p were also separated on the gel for immunoblotting as indicated.

Figure 7

Ultrathin cryosections of vesicles from reaction 1 (A and B) performed with Sec23/Lst1p and Sec23/24p (10 μg/ml each), or from reaction 2 (C and D) performed with Sec23/24p only (20 μg/ml). In both conditions, lst1-null SICs were used as donor membranes. Sections were probed with either anti-Lst1p (A and C) or anti-Sec24p (B and D), followed by protein A-gold (10-nm gold). The quantitative evaluation of the respective immunolabelings is shown in Table . Bar, 0.2 μm.