We first tested whether Der1p can self-associate, as demonstrated for its mammalian homologs. To this end, we generated two differently tagged versions of Der1p. The first construct contained at the C-terminus both S-peptide- and protein A-tags (Der1-SA), which interact with a speficic S-peptide binding protein and IgG, respectively. The second construct contained three hemagglutinin (HA) tags at the C-terminus of Der1p (Der1-3HA). As demonstrated previously, both constructs support ERAD, although they are not fully functional; nevertheless, they associate with all components of the Hrd1p complex [3
]. Haploid yeast cells expressing the two constructs under their endogenous promoters were mated to obtain diploid cells expressing both proteins at the same time. The cells were broken and cell lysates were either subjected directly to SDS-PAGE (input), or the lysate was incubated with beads containing the S-peptide binding protein, and bound proteins were analyzed by SDS-PAGE (IP). In both cases, the proteins were transferred to nitrocellulose and probed with IgG or HA antibodies, followed by peroxidase-coupled secondary antibodies. As shown in , lane 8, pull-down of Der1-SA co-isolated Der1-3HA. No band was seen with control cells that only expressed Der1-3HA (lane 7). These data indicate that Der1p can self-associate, consistent with results obtained with the mammalian Derlins.
Der1p and Dfm1p self-associate, but do not interact with each other
Next, we performed similar experiments with Dfm1p. In this case, the protein was fused at the C-terminus to a calmodulin peptide and a protein A module (Dfm1-CA) or to three HA tags (Dfm1-3HA). Cell lysates were incubated with calmodulin-coupled beads and bound proteins were analyzed by blotting with HA antibodies (, lane 4). Two bands were visible, one corresponding to the co-precipitated Dfm1-3HA protein (arrow head), the other to Dfm1-CA (star), which reacts with the IgG of the secondary antibodies. No bands were seen with control cells expressing only Dfm1-3HA (lane 3). These data thus show that Dfm1p, like Der1p, can form oligomers that contain at least two Dfm1p molecules.
Finally, we tested whether Der1p interacts with Dfm1p. Dfm1-CA was co-expressed with Der1-3HA in haploid yeast cells, and pull-down experiments were performed with calmodulin-containing beads. Despite the fact that both proteins could be detected in the input fractions (, lanes 2 and 6) and that Dfm1-CA was efficiently recovered in the bound fraction (lane 4), there was no co-isolation of Der1-3HA (lane 8). Thus, Der1p and Dfm1p do not appear to interact with one another.
Based on the observation that the Der1p and Dfm1p complexes appear to be distinct, we reasoned that they might contain different components. To identify interaction partners of Der1p and Dfm1p, we expressed Der1-SA and Dfm1-CA under their endogenous promoters. The yeast cells were broken and a membrane fraction was solubilized in 1% digitonin. The extract was incubated with IgG coupled to magnetic beads, and bound proteins were eluted with SDS. They were separated by SDS-PAGE and stained with Coomassie blue. A control, performed with wild type cells containing untagged proteins, showed that the major bands eluted from the beads were IgG heavy and light chains, which presumably were not covalently bound (, lane 1–3). However, with both Der1-SA amd Dfm1-CA a number of additional bands were seen (numbered in lanes 2 and 3). The identity of the bands was determined by mass spectrometry (). We also analyzed the proteins bound to the IgG beads directly by mass spectrometry after precipitation with trichloroacetic acid ().
Interaction partners of Der1p and Dfm1p
Table Summary of interacting proteins found when immunoprecipitation was performed using tagged Der1p (Der1-SA) or tagged Dfm1p (Dfm1-CA). The bound proteins were identified using mass spectrometry. We analyzed either the total protein eluate after precipitation (more ...)
All Der1p interaction partners have been identified before: Cdc48p (band 1), Usa1p and Hrd3p (band 2), Hrd1p, Ubx2p, Yos9p, and Npl4p (band 3), Der1p-TAP and Ufd1p (band 5); band 4 contained only rabbit IgG. The membrane proteins Hrd1p, Hrd3p, Usa1p, and Der1p are members of the Hrd1p complex, Npl4p and Ufd1p are cofactors of the Cdc48p ATPase, Ubx2p is a membrane-bound binding partner of Cdc48p, and Yos9p is a luminal lectin-like chaperone. For all proteins there is evidence that they participate in ERAD.
The majority of the Dfm1p components are identical to those in the Der1p complex: Cdc48p (band 1), Usa1p and Hrd3p (band 2), Yos9p and Ubx2p (band 4), and Npl4p and Hrd1p (band 5). The approximate levels of most of these proteins was about the same in the two complexes, as judged from the number of peptides identified, with the exception of Cdc48p, which was more abundant in the Dfm1p complex (see band 1 in lanes 2 and 3). Dfm1p-TAP was contained in band 6 that was dominated by IgG. In addition to these proteins, we found Kar2p (band 3) as well as Ubx1p and Ubx7p (band 6). Kar2p (BiP) is a member of the Hsp70 family of chaperones and is an abundant luminal ER protein. The detection of Ubx1p and Ubx7p is particularly interesting because both proteins are established cofactors of the Cdc48p ATPase, but have no known function in ERAD. Bands 7 and 8 contained peptides of Dfm1p and Cdc48p, respectively. These could originate from proteolysis or, in the case of Dfm1p, represent untagged Dfm1p. In contrast to the results with the Der1p complex, Ufd1p was undetectable in the Dfm1p complex. The results were the same when the samples were analyzed directly after trichloroacetic acid precipitation (), indicating that no major component was missed in the Coomassie-stained gel.
To confirm that the Der1p- and Dfm1p- complexes contain distinct cofactors of the Cdc48p ATPase, we performed pull-down experiments with tagged proteins. Der1-3HA was expressed together with Ubx1p, Ufd1p, or Ubx7p, all containing at their C-termini both S-peptide- and protein A- tags (Ubx1-SA, Ufd1-SA, Ubx7-SA). All proteins were expressed under their endogenous promoters. The expression level of Ubx7p-SA was lower than that of Ubx1p-SA or Ufd1p-SA, as determined by SDS-PAGE followed by probing with IgG (, lower panel, lane 3 versus lanes 1 and 2). All SA-tagged proteins were also efficiently recovered with beads to which the S-peptide binding protein had been coupled (lanes 5–7). When probed with HA antibodies for co-precipitation of Der1-HA, a band was only seen in pull-down experiments with Ufd1-SA (, upper panel, lane 6; arrow head). This band was not seen in the absence of the SA-tagged proteins (lane 8). No association of Der1-3HA was observed with either Ubx1-SA or Ubx7-SA (lanes 5 and 7) (the upper bands in lanes 5–7 result from the detection of the protein A moiety in the SA-tagged protein by the secondary antibody and serve as an additional control for the recovery of the tagged proteins). These data show that Ufd1p, but not Ubx1p or Ubx7p, is associated with the Der1p complex, in agreement with the mass spectrometry experiments. It should be noted that in the two sets of experiments, the pull-down was performed in a reciprocal manner (Ufd1-SA in versus Der1-SA in ), confirming the existence of a genuine complex.
Der1p and Dfm1p bind a different set of Cdc48p cofactors
Similar experiments were performed with the Dfm1p complex. In this case, Dfm1-3HA was co-expressed with SA-tagged versions of Ubx1p, Ufd1p, or Ubx7p, and the SA-tagged proteins were bound to magnetic beads containing coupled IgG. When the bound proteins were probed with HA antibodies, a major Dfm1-3HA band was seen when the pull-down was performed with Ubx1-SA (, upper panel, lane 5; arrow head) (the upper band results from detection of the protein A moiety in the SA-tagged protein). Smaller quantities of Dfm-3HA bands were pulled down by Ufd1-SA or Ubx7-SA (upper panel, lanes 6 and 7). Whereas Ufd1-SA was expressed at about the same level as Ubx1-SA (see lower panel, lanes 1 and 2), the level of Ubx7-SA was significantly lower (lane 3). Thus, the Dfm1p complex contains a significantly higher fraction of Ubx7p than Ufd1p. These results again confirm our mass spectrometry data, as they indicate that, in contrast to Der1p, Dfm1p is associated with Ubx1p and Ubx7p. Although small amounts of Ufd1p were associated with Dfm1p in the pull-down experiments, the amounts of Ufd1p in the Der1p complex were significantly higher (compare the ratio of the Der1-3HA band to the Ufd1-SA band in , upper panel, lane 6 with the ratio of these bands in , upper panel, lane 6). The low level of Ufd1p in the Dfm1p complex accounts for the lack of Ufd1p peptides in the mass spectrometry experiments.