Emp46p forms a complex with Emp47p in the ER. (A) KSY062 (sec12-4, HA-EMP46) cells were incubated at 32°C for 120 min before microsome preparation, and microsomes (75 mg) were incubated in the presence (+) or absence (-) of DSP. HA-Emp46p was immunoprecipitated from the Triton X-100–solubilized membrane with the anti-HA antibody. Samples were separated by SDS-PAGE under a reducing condition and visualized by Coomassie staining. Specific protein bands were excised and trypsin-digested, and the resulting peptide mixtures were analyzed by MALDI mass spectrometry. Identified proteins are indicated on the right. IgG-LC, IgG light chain; asterisks, partial proteolytic breakdown products of indicated proteins. (B) Microsomes from emp46Δemp47Δ cells expressing myc-Emp47p and HA-Emp46p were solubilized with 0.05% n-dodecyl maltoside. The resulting extract was cleared by centrifugation and immunoprecipitated (IP) with the antimyc antibody followed by immunoblotting with the antimyc or anti-HA antibody. Total lane (T) represents 5% of the starting microsomal extract.

Emp46p is associated with Emp47p through the coiled-coil region in its luminal domain, which dissociates in the Golgi. (A) Diagram of constructs encoding Emp47p and Emp46p derivatives. Emp47p derivatives are N-terminally tagged with myc and GFP (Δ383, Δ333, and Δ281) or 2× myc (Δ281–333). Emp46p-Δ281–333 is tagged by HA. (B) Immunoblots of anti-HA native immunoprecipitations from solubilized microsomes expressing HA-Emp46p with Emp47p-Δ383, -Δ333, -Δ281, and -Δ281–333. Total lanes (T) represent 5% of immunoprecipitation (IP) lanes. (C) Solubilized microsomes expressing myc-Emp47p and HA-Emp46p-Δ281–333 were subjected to native immunoprecipitation with antimyc antibody as above. (D) ER and Golgi fractions from sucrose density gradient in Figure 2B (ER, fraction 8; Golgi, fraction 4) and microsome-derived COPII vesicles were solubilized, and Emp47p-Emp46p complexes were detected by native immunoprecipitation as in Figure 1B. ER-Golgi marker, Sed5p, is shown as a negative control.

Emp47p oligomerization acts as a signal for its incorporation into COPII vesicles but is not required for COPII binding. (A) COPII vesicle budding from emp46Δemp47Δ cells expressing Emp47p-Δ281–333 with GFP-Emp47p (left) or HA-Emp46p (right) microsomes as in Figure 2A. (B) Microsomes prepared from cells expressing myc-Emp47p and HA-Emp46p (left) or myc-Emp47p-Δ281–333 and HA-Emp46p (right) were incubated with GST-Sar1p and the purified Sec23/24p complex in the presence of GMP-PNP or GDP-βS. Subsequently, digitonin-soluble prebudding complexes were recovered on glutathione beads. Emp46p, Emp47p, Sec22p, and Sec61p in the prebudding complex were detected by immunoblotting. Total (T) represents 0.5% of the total solubilized membranes.

Emp47p is required for the ER exit of Emp46p. (A) In vitro budding reactions with microsomes prepared from emp46Δemp47Δ cells expressing both myc-Emp47p and HA-Emp46p (Wild-type), or either HA-Emp46p (emp47Δ) or myc-Emp47p (emp46Δ). Ten percent of the total reaction (T) and budded COPII vesicles isolated after incubation with (+) or without (-) COPII proteins were analyzed by SDS-PAGE followed by immunoblotting with the anti-HA or antimyc antibody. Sec61p as a negative control and Sec22p as a positive control were detected with polyclonal antibodies. (B) Subcellular distribution of Emp46p in the presence or absence of Emp47p. Whole cell lysates from indicated strains were separated on 20–60% sucrose density gradient. Relative levels of Pho8p (vacuole marker), myc-Emp47p (Golgi marker), HA-Emp46p, and Sec61p (ER marker) in each fraction were quantified by densitometry of immunoblots. (C) Expression level of Emp47p influences the subcellular distribution of Emp46p.

Emp47p alone forms oligomers through its coiled-coil region in the ER, whereas Emp46p alone is present as a monomer. (A and B) Microsomes from emp46Δemp47Δ cells expressing differently tagged Emp47p (a, myc-GFP-Emp47p and myc-Emp47p) and Emp46p (b, GFP-HA-Emp46p and HA-Emp46p) were solubilized and applied to native immunoprecipitation as in Figure 1B, followed by immunoblotting with antimyc (a) or anti-HA (b). (C) emp46Δemp47Δ cells expressing indicated proteins were visualized by confocal laser microscopy. Bars, 5 μm. (D) Subcellular localization of Emp47p deletion constructs shown in Figure 3A coexpressed with wild-type Emp47p. (E) Anti-myc immunoblots of anti-GFP native immunoprecipitation from solubilized microsomes expressing myc-GFP-Emp47p and myc-Emp47p-Δ281–333 as in A and B. Total (T) represents 5% of the immunoprecipitation lane.

Emp47p forms a large oligomeric complex. ER and Golgi fractions were prepared from emp46Δemp47Δ cells expressing indicated proteins by sucrose density gradient as in Figure 2B (ER, fraction 8; Golgi, fraction 4), and solubilized with 0.05% n-dodecyl maltoside. Extracts were applied to a G3000SWXL gel filtration column. Eluates were subjected to SDS-PAGE followed by immunoblotting using antimyc and anti-HA antibodies. Molecular size standards used are thyroglobulin (669 kDa), aldolase (158 kDa), ovalbumin (43 kDa), and ribonuclease A (13.7 kDa).