BAT3 guides misfolded glycoproteins out of the endoplasmic reticulum

Abstract

Secretory and membrane proteins that fail to acquire their native conformation within the lumen of the Endoplasmic Reticulum (ER) are usually targeted for ubiquitin-dependent degradation by the proteasome. How partially folded polypeptides are kept from aggregation once ejected from the ER into the cytosol is not known. We show that BAT3, a cytosolic chaperone, is recruited to the site of dislocation through its interaction with Derlin2. Furthermore, we observe cytoplasmic BAT3 in a complex with a polypeptide that originates in the ER as a glycoprotein, an interaction that depends on the cytosolic disposition of both, visualized even in the absence of proteasomal inhibition. Cells depleted of BAT3 fail to degrade an established dislocation substrate. We thus implicate a cytosolic chaperone as an active participant in the dislocation of ER glycoproteins.

Figure 1. BAT3 associates with Derlin2.

A. HA-Ri332 was synthesized in a rabbit reticulocyte lysate in the presence or absence of canine pancreatic microsomal membranes (MM). Following NP40-mediated lysis, Ri332 was retrieved by immunoprecipitation. The immunoprecipitate and input samples were blotted for either BAT3 or HA as indicated. B. 293T cells were subjected to NP40 lysis, followed by retrieval of the indicated proteins. Pre-immune serum served as a control. The eluates were blotted for BAT3, as were the input control samples. C. Immunofluorescence of Hela cells using antibodies against endogenous BAT3 (green) and Derlin2 (red). Scale bar = 5 µm. D. 293T cells were transiently transfected with the indicated constructs, subjected to NP40 lysis followed by an immunoprecipitation for Derlin2. Immunoprecipitates were blotted for BAT3. Input samples were blotted for BAT3 and Derlin2. The asterisk indicates non-specific polypeptides.

Figure 2. BAT3 localizes to a complex with Derlin2 and Ri332.

Hela cells were plated on coverslips and transiently transfected with HA-Ri332 and empty vector (A), YOD1 C160S (B) or UBX-EBV (C). After paraformaldehyde fixation, cells were labeled for HA (green), BAT3 (red), and Derlin2 (blue). Scale bar = 5 µm.

Figure 3. BAT3 is required for dislocation of TCRα.

A. 293T were stably transduced with short RNA hairpins against either GFP or BAT3. SDS lysates were immunoblotted for either BAT3 or p97. B. BAT3 knock-down cells (a) were transiently transfected with HA-Ri332 and subjected to pulse-chase analysis. Densitometric quantitation of the relative amount of protein is shown (n = 9). C. As in (b), except that cells were transfected with TCRα (n = 3).

Figure 4. Impaired BAT3 recruitment to Derlin2 slows dislocation.

A. 293T cells were transiently transfected with TCRα and either pcDNA, Derlin2, or Derlin2-GFP (see figure 1D). TCR degradation was assessed by pulse-chase analysis. B. Densitometric quantitation of the relative amount of protein is shown (n = 4).

Figure 5. TCRα is engaged by BAT3.

293T cells were transiently transfected with TCRα and either empty vector, UBX-EBV, or p97 QQ, and labeled overnight with [³⁵S] methionine/cysteine to achieve steady state labeling. Cells were harvested and subjected to NP40 lysis. The lysates were precleared using pre-immune serum and immobilized protein A. Lysates were adjusted for total levels of incorporated isotope and subjected to immunoprecipitation for TCRα. The captured protein was eluted in 1% SDS at 37°C followed by a second immunoprecipitation for the indicated proteins.