Boca-dependent maturation of beta-propeller/EGF modules in low-density lipoprotein receptor proteins

Abstract

The extracellular portions of cell surface receptor proteins are often comprised of independently folding protein domains. As they are translated into the endoplasmic reticulum (ER), some of these domains require protein chaperones to assist in their folding. Members of the low-density lipoprotein receptor (LDLR) family require the chaperone called Boca in Drosophila or its ortholog, Mesoderm development, in the mouse. All LDLRs have at least one six-bladed beta-propeller domain, which is immediately followed by an epidermal growth factor (EGF) repeat. We show here that Boca is specifically required for the maturation of these beta-propeller/EGF modules through the secretory pathway, but is not required for other LDLR domains. Protein interaction data suggest that as LDLRs are translated into the ER, Boca binds to the beta-propeller. Subsequently, once the EGF repeat is translated, the beta-propeller/EGF module achieves a more mature state that has lower affinity for Boca. We also show that Boca-dependent beta-propeller/EGF modules are found not only throughout the LDLR family but also in the precursor to the mammalian EGF ligand.

Figure 1

Schematic representation of YWTD β-propeller-containing proteins. Most proteins with this type of β-propeller belong to the LDLR family, which also have type A and EGF repeats in their extracellular domains. Shown here are Drosophila LpR2 and Arrow and human LDLR and LRP1. The non-LDLR proteins with YWTD β-propellers used in this work are Drosophila Nidogen, an extracellular matrix protein, the tyrosine kinase receptor Sevenless, and mouse EGFP. The yellow bars in the Nidogen β-propeller represent two disulfide bonds. Sevenless has a cytoplasmic tyrosine kinase domain (TyrK) and fibronectin type III modules (F) in the extracellular region. The red brackets indicate the domains analyzed in this work. Arrow β-propeller/EGF pairs are numbered from N to C. The domain structure for Drosophila Nidogen was tentatively derived from a predicted open reading frame.

Figure 2

Flow diagram of the two assays used to analyze the trafficking of LpR2 derivatives. Both assays begin with expression of HA-tagged LpR2 derivatives into wild-type (wt) and boca⁻ S2 cells. In assay 1, the cells were transfected with LpR2 derivatives that contain a TM and examined by immunofluorescence and confocal microscopy. The cells were stained to detect Boca (blue), which is a marker for the ER (Culi and Mann, 2003), the LpR2 derivative (green, using the HA epitope), and F-actin (red), which serves as a marker for the cell membrane. For clarity, the green channel is shown to the right of the merged image. In wt S2 cells, LpR2 is found in intracellular vesicles and at the cell membrane, which appears yellow due to colocalization with F-actin (arrows). In boca⁻ S2 cells, LpR2 is found diffusely throughout the cytoplasm and not at the cell membrane, which appears red due to lack of colocalization with F-actin. This staining is consistent with LpR2 accumulating in the ER, which is diffuse in S2 cells as shown by the distribution of the ER marker Boca in wild-type cells (blue) (see also Culi and Mann, 2003). In assay 2, the cells were transfected with LpR2 derivatives that do not have a TM. Transfection efficiencies were normalized by cotransfecting with a lacZ expression plasmid and comparing β-galactosidase activities in the cell extracts. The amount of the HA-tagged protein in the media and in total cell lysates was examined by immunoblotting with an anti-HA antibody. Although LpR2 was detected intracellularly in both wild-type and boca⁻ S2 cells, it was only secreted from wild-type cells.

Figure 3

The β-propeller/E3 module constitutes the minimal Boca-dependent fragment of LpR2. (A) Assay 1: Full-length LpR2 and several deletion derivatives were transfected into wild-type (wt) and boca⁻ S2 cells. A schematic representation of the various LpR2 derivatives is shown on the left. The seven A type repeats (A), three EGF repeats (E1, E2, and E3), β-propeller (β), O-glycosylation sequence (Gly), and TM are indicated. The domains listed refer to those present in the protein being analyzed. Cells were stained as described in Figure 2. In these images, the white arrows point to membrane localization of the HA-tagged protein. Of these proteins, only LpR2_Gly, LpR2_A, and LpR2_E1/E2 show membrane localization in the absence of Boca. (B) Assay 2: Proteins without a TM domain were expressed in wild-type (+) and boca⁻ (−) cells. Secreted and intracellular fractions were analyzed by immunoblotting with anti-HA antibody. The domains listed refer to those present in the protein being analyzed. Of these proteins, only LpR2 and LpR2_β/E3 require Boca for secretion into the media.

Figure 4

Boca interacts with the β-propeller. (A) The LpR2ΔTM derivatives HA-β, E3-HA, and mock DNA were cotransfected into wild-type (+) and boca⁻ (–) S2 cells as indicated. Twice the amount of E3-HA DNA was used compared to HA-β. Secreted and intracellular fractions were analyzed by immunoblotting with anti-HA antibody. The HA-β and E3-HA bands are indicated at the right of the figure. Note that for the intracellular fraction, multiple glycosylation bands can be detected for each protein. Secretion of E3-HA is strongly impaired by the coexpression of HA-β, both in wild-type and boca⁻ cells. (B) The indicated LpR2ΔTM derivatives were expressed in wild-type S2 cells, the cells treated with a crosslinking reagent, and cell extracts immunoprecipitated with anti-HA antibody. An experiment carried out with untransfected cells (–) represents the level of background observed in this experiment. The TM protein Frizzled2 (Fz2), whose trafficking is Boca independent (Culi and Mann, 2003), served as another negative control. The immunoprecipitates were immunoblotted with anti-HA and anti-Boca in parallel as indicated. Of these proteins, only LpR2_β co-immunoprecipitated Boca above background. (C) LpR2_β^W530K/E3, which has a mutation in Trp530 of LpR2, co-immunoprecipitated Boca. In contrast, wild-type LpR2_β/E3 and LpR2 failed to co-immunoprecipitate Boca. All proteins used in this experiment are ΔTM derivatives. (D) Arrow (Arr), but not LpR2 or Fz2, co-immunoprecipitated Boca. All proteins used in this experiment contain a TM.

Figure 5

β-Propeller/EGF modules from other LDLRs also require Boca. (A) All four β-propeller/EGF pairs from Arrow and the seventh β-propeller/EGF pair from human LRP1 (hLRP1(7)) were tested for Boca dependency using the secretion assay. LpR2_β/E3 was included as a positive control. All proteins required Boca except for Arr2, for which Boca dependency could not be assessed because it failed to be secreted even in wild-type (+) cells. (B) Without their C-terminal EGF repeats, the β-propellers from LpR2 and hLRP1(7) failed to be secreted in wild-type and boca⁻ cells.

Figure 6

A β-propeller/EGF pair from EGFP requires Boca. (A, B) β-Propellers/EGF pairs (β/EGF) (A) and β-propellers from Sevenless (Sev), murine EGF precursor (mEGFP), and Nidogen were tested in S2 cells for Boca-dependent maturation using the secretion assay. Nidogen^Cys* denotes a β/EGF protein that is unable to form its two intrapropeller disulfide bridges. Of these proteins, only the β-propeller/EGF pair from EGFP required Boca for secretion. (B) Clones of homozygous boca¹ cells were induced in the eye imaginal disc and stained for Sevenless (red) and GFP (green). The absence of GFP marks the boca¹ mutant cells. The focal plane was limited to the apical surface of these cells. Sevenless was localized at the apical cell membrane in both boca⁺ and boca¹ cells.

Figure 7

Boca and Mesd are functionally equivalent in S2 cells. Constructs encoding a secreted version of LpR2 or the β/E3 module from LpR2 were transfected into wild-type (+) and boca⁻ S2 cells with a Mesd expression construct or mock DNA as indicated. Secreted and intracellular levels of the HA-tagged LpR2 proteins were measured using assay 2. For both proteins, secretion was rescued in boca⁻ cells by expressing Mesd.