Article Figures & Data
Figures
- Figure 1.
BiP and Calreticulin Form an ATP-Dependent Complex in the Absence of Stress.
(A) Tobacco protoplasts were labeled in vivo for 3 hr under normal culture conditions (C) or at 37°C during heat shock treatment (H). The gel at left shows labeled proteins that were immunoprecipitated with anti-calreticulin antiserum (anti-cal) and analyzed by SDS-PAGE. The gel at right shows the proteins selected with anti-BiP antiserum from the supernatant resulting from washing of the immunoprecipitate with BiP release buffer containing 3 mM ATP. Molecular mass markers are given at right in kilodaltons. The positions of BiP (75 kD) and calreticulin (Cal; 60 kD) are indicated at left.
(B) Immunoprecipitation of the protoplast extract labeled in vivo was performed with anti-calreticulin antiserum, and the immunoprecipitate was washed with either BiP release buffer alone (C) or BiP release buffer containing adenosine nucleotides, as indicated, at a concentration of 3 mM.
- Figure 2.
The BiP–Calreticulin Complex Is Stable.
(A) Tobacco protoplasts were labeled in vivo for 1 hr followed by a chase of 1, 2, 4, and 8 hr (as indicated for each lane). Extracts were immunoprecipitated with the anti-calreticulin antiserum, separated by SDS-PAGE, and then quantified using a PhosphorImager. The histogram shows the signal strength of both calreticulin (open bars) and BiP (diagonally striped bars) during the chase as a percentage of the calreticulin signal at time 0 hr. The positions of BiP and calreticulin (Cal) are indicated at left on the autoradiograph.
(B) Tobacco protoplasts were labeled in vivo for 1 hr and extracted immediately (0 hr) or after an 8-hr chase, as indicated above the lanes. Lanes 1 and 5 show proteins immunoprecipitated with anti-BiP antibodies. Lanes 2 and 6 show proteins immunoprecipitated by anti-BiP antibodies from the supernatant of immunoprecipitations with anti-calreticulin antibodies. Lanes 3 and 7 show proteins immunoprecipitated with anti-calreticulin antibodies. Lanes 4 and 8 are controls showing background bands that precipitate nonspecifically from BiP and calreticulin-depleted extracts. Molecular mass markers are indicated at right in kilodaltons.
- Figure 3.
Interaction of BiP with Calreticulin and with Unassembled Phaseolin.
Protoplasts from the leaves of transgenic plants expressing Δ363 phaseolin were labeled in vivo for 1 hr. Immunoprecipitation was performed with anti-phaseolin, anti-BiP, or anti-calreticulin antibodies, as indicated. Immunoprecipitates were then washed with BiP release buffer alone (−) or supplemented with 3 mM ATP (+). Molecular mass markers are given at right in kilodaltons. At left, the positions of BiP, calreticulin (Cal), and phaseolin (Phas) are indicated. Phaseolin was synthesized in two glycoforms having molecular masses of 46 and 43 kD. Note that more BiP coprecipitates with calreticulin than with the overproduced assembly-defective phaseolin.
- Figure 4.
Effect of Low pH and Cation Chelators on Complex Dissociation.
(A) Immunoprecipitations were performed with anti-calreticulin antibodies, and the resulting pellets were washed for 30 min with buffers ranging from pH 7.5 to 4.5, as indicated above the lanes. The level of complex association seen after washing with the various buffers is shown in the histogram. The degree of association seen after washing at pH 7.5 was defined as 100%. The positions of BiP and calreticulin (Cal) are indicated at left. Notice that low pH significantly reduces complex stability.
(B) Immunoprecipitations were performed with anti-calreticulin antibodies in the presence of NET gel buffer alone (diagonally striped bar), with an additional 1 or 10 mM EDTA (open bars), or 1 or 10 mM EGTA (horizontally striped bars). The degree of complex association observed after immunoprecipitation is shown in the histogram. The level of association seen in the control sample (C) was defined as 100%. The positions of BiP and calreticulin are indicated at left.
- Figure 5.
Complex Formation in Calreticulin or BiP Overproducers.
(A) Immunoprecipitations were performed with extracts from in vivo–labeled protoplasts prepared from control plants, calreticulin overproducers (calret+), and BiP overproducers (BiP+), as indicated above the lanes. Diluted extracts were used for calreticulin overproducers to immunoprecipitate a similar amount of labeled calreticulin. Two independent experiments are shown, and the ratio of BiP to calreticulin (cal) is given in the histogram. The ratio in the first control lane was defined as 100%. Note that this ratio is much lower in calreticulin overproducers, indicating that no additional BiP is recruited into the complex. Note also the lower molecular mass band of calreticulin in extracts from BiP overproducers (arrowhead). The positions of BiP and calreticulin (Cal) are indicated at left.
(B) Immunoprecipitations were performed using anti-calreticulin antibodies on the same in vivo–labeled BiP overproducer extract as described in (A), followed by incubation in the presence (+) or absence (−) of endoglycosidase H (endo H). At left, the positions of the glycosylated and deglycosylated forms of calreticulin are indicated together with that of the calreticulin species seen only in the BiP overproducers (new calret). Molecular mass markers are given at right in kilodaltons.
- Figure 6.
Complex Formation with HDEL-less BiP.
Immunoprecipitations were performed with in vivo–labeled protoplast extracts from untransformed plants (BiP) and from transgenic tobacco plants producing a mutant BiP lacking eight C-terminal amino acids (ΔBiP). The antiserum used is indicated above the lanes. Note that the ratio of BiPΔHDEL to BiP is unaltered when coprecipitated with calreticulin. The positions of wild-type BiP (BiP), BiP lacking the retention signal (BiPΔHDEL), and calreticulin (Cal) are given at left. Molecular mass markers are given at right in kilodaltons.
- Figure 7.
BiP and Calreticulin Form Large Protein Complexes.
(A) Five hundred microliters of a 2.5 mg/mL extract of suspension-cultured tobacco cells was separated on a Superose 12 column using a Pharmacia fast-protein liquid chromatography system. The resulting fractions were then analyzed by protein gel blotting, using both anti-BiP and anti-calreticulin antisera. The positions of BiP and calreticulin (Cal) are indicated at left. The estimated molecular masses of the complexes formed by BiP and calreticulin are >400 (P1), 300 (P2), 140 (P3), and 70 kD (P4). The latter corresponds well with the monomeric forms of BiP and calreticulin.
(B) The indicated peak fractions from (A) were individually pooled, and 50 μL of pools P1 and P2 were immunoprecipitated with anti-calreticulin antibodies and 10 μL of protein A–Sepharose gel. To avoid saturation of the anti-calreticulin antiserum, only 10 μL was used for P3 and P4. The pellets obtained were then washed in 30 μL of BiP release buffer in the presence (+) or absence (−) of 3 mM ATP to release coprecipitated material. Twenty microliters of clear supernatant was then analyzed by protein gel blotting using anti-BiP antibodies representing 50% of the total material. Ten microliters of protein A–Sepharose gel and 10 μL of supernatant were not added to avoid contamination by antibodies. The control lane (C) was loaded with 20 μL of starting material (fraction P1) for comparative purposes. Note that the intensity of the BiP bands in P1 and P2 is comparable to that of the control lane, which would be expected if most of the BiP in these fractions is associated with calreticulin. The intensity of the BiP band in P3 is lower, but this is because five times less material was used for the immunoprecipitation (see above). The position of BiP is indicated at left.
- Figure 8.
Model for BiP–Calreticulin Interaction.
A schematic representation of the interaction of BiP with assembly-defective phaseolin (Phas) and with calreticulin (Calret). The antibody binding sites of BiP remain exposed when bound to phaseolin but are masked by the interaction with calreticulin. This indicates that a different portion of the BiP molecule is involved in the interaction.
