Involvement of the penta-EF-hand protein Pef1p in the Ca2+-dependent regulation of COPII subunit assembly in Saccharomyces cerevisiae

Abstract

Although it is well established that the coat protein complex II (COPII) mediates the transport of proteins and lipids from the endoplasmic reticulum (ER) to the Golgi apparatus, the regulation of the vesicular transport event and the mechanisms that act to counterbalance the vesicle flow between the ER and Golgi are poorly understood. In this study, we present data indicating that the penta-EF-hand Ca(2+)-binding protein Pef1p directly interacts with the COPII coat subunit Sec31p and regulates COPII assembly in Saccharomyces cerevisiae. ALG-2, a mammalian homolog of Pef1p, has been shown to interact with Sec31A in a Ca(2+)-dependent manner and to have a role in stabilizing the association of the Sec13/31 complex with the membrane. However, Pef1p displayed reversed Ca(2+) dependence for Sec13/31p association; only the Ca(2+)-free form of Pef1p bound to the Sec13/31p complex. In addition, the influence on COPII coat assembly also appeared to be reversed; Pef1p binding acted as a kinetic inhibitor to delay Sec13/31p recruitment. Our results provide further evidence for a linkage between Ca(2+)-dependent signaling and ER-to-Golgi trafficking, but its mechanism of action in yeast seems to be different from the mechanism reported for its mammalian homolog ALG-2.

Figure 1. Ca²⁺-free form of Pef1p binds to the Sec13/31p complex.

(A) Sequence alignment of the penta-EF-hand motifs of S. cerevisiae Pef1p (residues 155–195 and 227–265) and mouse ALG-2 (residues 24–61 and 93–127) obtained by the CLUSTALW program. Highlighted residues (white letters on black) indicate amino acids that are identical to those of ALG-2, and grey residues indicate conservation. Arrows indicate the critical calcium interacting residues E180, E181, and E248. (B) Sec13/31p (80 nM) was incubated with 40 nM of GST, GST-Pef1p, or GST-Pef1p-E180/181/248A in the presence of EGTA (5 mM) or CaCl₂ (1 mM) as indicated. The reactions were mixed with glutathione-Sepharose beads. After extensive washing, bound proteins were eluted and analyzed by SDS-PAGE and immunoblotting with anti-Sec31p antibody.

Figure 2. The proline-rich region of the Sec31p subunit is involved in the interaction of the Sec13/31p complex with Pef1p.

(A) Schematic representation of the domain structure of Sec31p and the MBP-Sec31p constructs used to identify the region in Sec31p required for Pef1p binding. Numbers indicate amino acid positions defining each deletion construct. (B) The truncated MBP-Sec31p constructs were incubated with GST or GST-Pef1p as indicated. The reactions were mixed with glutathione-Sepharose beads. After extensive washing, bound proteins were eluted and analyzed by SDS-PAGE and immunoblotting with anti-MBP antibody. Asterisks denote the positions of the MBP-Sec31p constructs.

Figure 3. Assembly of the COPII coat onto liposomes in the presence of Pef1p.

The reaction initially contained liposomes (100 µg/mL), Sar1p (950 nM), and GMP-PNP (0.1 mM) in the presence (closed circles) or absence (open circles) of GST-Pef1p (520 nM) (A) or GST-Pef1p-E180/180/248A (520 nM) (B). EGTA (5 mM) was included in (A). After preincubation, Sec23/24p (160 nM) and Sec13/31p (260 nM) were added at the indicated time points, and the light scattering of the suspension was monitored.

Figure 4. GAP stimulation activity of Sec31p in the presence of Pef1p.

The reaction initially contained liposomes (50 µg/mL), Sar1p (500 nM), GTP (0.1 mM), and either EGTA (5 mM) (A) or CaCl₂ (1 mM) (B) in the presence of Sec13/31p (50 nM) (open squares) or both Pef1p (70 nM) and Sec13/31p (50 nM) (closed circles). After preincubation, Sec23/24p (160 nM) was added at the indicated time point. Transition of Sar1p from the GTP-bound to the GDP-bound state was monitored by tryptophan fluorescence of Sar1p at 340 nm.

Figure 5. Pef1p overexpression causes defects in yeast cell growth, but does not affect anterograde transport.

(A) Isogenic wild-type (BY4741) or pef1Δ cells transformed with GAL1-PEF1 constructs or vector (pYES2) as indicated were grown to saturation at 30°C, adjusted to an OD₆₀₀ of 0.5, and 5 µL of a 10-fold dilution series was spotted onto selective plates containing 2% glucose (left panel) or 2% galactose (right panel). (B) Wild-type and pef1Δ cells transformed with the same plasmids as in (A) were grown at 30°C to log phase in selective media supplemented with 2% glucose (left panel) or 2% galactose (right panel) to induce overexpression of the indicated Pef1p forms. Equal amounts of whole cell extracts were subjected to immunoblotting with anti-CPY antibody. The erv29Δ strain was used as a control. The ER (p1) and mature (m) forms of CPY are indicated.

Figure 6. Cellular localization of Pef1p.

Log-phase cultures of pef1Δ mutant cells co-expressing Sec13p-mCherry and either GFP-Pef1p (A) or GFP-Pef1p-E180/181/248A (B) were visualized by confocal microscopy. The panels on the right show merged fluorescence images. Scale bars, 5 µm.