A Quantitative Model for BicD2/Cargo Interactions

Abstract

Dynein adaptor proteins such as Bicaudal D2 (BicD2) are integral components of the dynein transport machinery, as they recognize cargoes for cell cycle-specific transport and link them to the motor complex. Human BicD2 switches from selecting secretory and Golgi-derived vesicles for transport in G1 and S phase (by recognizing Rab6^GTP), to selecting the nucleus for transport in G2 phase (by recognizing nuclear pore protein Nup358), but the molecular mechanisms governing this switch are elusive. Here, we have developed a quantitative model for BicD2/cargo interactions that integrates affinities, oligomeric states, and cellular concentrations of the reactants. BicD2 and cargo form predominantly 2:2 complexes. Furthermore, the affinity of BicD2 toward its cargo Nup358 is higher than that toward Rab6^GTP. Based on our calculations, an estimated 1000 BicD2 molecules per cell would be recruited to the nucleus through Nup358 in the absence of regulation. Notably, RanGTP is a negative regulator of the Nup358/BicD2 interaction that weakens the affinity by a factor of 10 and may play a role in averting dynein recruitment to the nucleus outside of the G2 phase. However, our quantitative model predicts that an additional negative regulator remains to be identified. In the absence of negative regulation, the affinity of Nup358 would likely be sufficient to recruit BicD2 to the nucleus in G2 phase. Our quantitative model makes testable predictions of how cellular transport events are orchestrated. These transport processes are important for brain development, cell cycle control, signaling, and neurotransmission at synapses.

Figure 1.

Purification of the cargo binding domain of BicD2 and its cargoes. (A) For biophysical studies, Nup358-RBD2 (black bar), a fragment of Nup358 that includes the BicD2 binding site and the two adjacent Ran binding domains (RBD red), was purified. (B) The N-terminal domain (NTD, orange) of BicD2 interacts with dynein/dynactin, while the CTD (blue) recruits cargo.^, For biophysical studies, the BicD2-CTD was purified (black bar). (C) In absence of cargo, the NTD and CTD of BicD2 form an autoinhibited state that cannot recruit dynein/dynactin.^,,, (D−F) SDS-PAGE analysis of purified proteins is shown. Masses of molecular weight standards in kDa are indicated on the left. (D) BicD2-CTD. (E) Nup358-RBD2. (F) Rab6^GTP. (G) Purification of a RanGTP/Nup358 complex. Purified Nup358-RBD2 and RanGTP were mixed. Nup358-RBD2 with RanGTP bound was separated by gel filtration. SDS-PAGE analysis of the elution fractions is shown.

Figure 2.

BicD2/cargo complexes predominantly form 2:2 oligomers.(A) The purified BicD2-CTD was analyzed by SEC-MALS (at 2 mg/mL). Rayleigh ratio (blue) and molar mass MW (red) versus the elution volume are shown. The determined molar mass is MW = 23.3 ± 1.3 kDa, which closely matches the mass of a BicD2 dimer (20.8 kDa). (B) Purified Nup358-RBD2/BicD2-CTD complex was analyzed by SEC-MALS (at 14 mg/mL). Refractive index (blue) and molar mass MW (red) versus the elution volume are shown. The determined molar mass is MW = 155.4 ± 7.8 kDa, which is slightly below the molar mass of a 2:2 complex of Nup358-RBD2/BicD2-CTD (174.6 kDa). (C) The purified Rab6^GTP_min was analyzed by SEC-MALS (at a concentration of 6 mg/mL). Rayleigh ratio (blue) and molar mass MW (red) versus the elution volume are shown. The determined molar mass is MW = 21.5 ±1.0 kDa, closely matching the molar mass of a Rab6^GTP_min monomer (19.0 kDa). (D) The Rab6^GTP/BicD2-CTD complex was analyzed at a protein concentration of 7 mg/mL by SEC-MALS. The molar mass of the major peak is MW= 61.0 ± 3 kDa, which is slightly less than the molar mass of a 2:2 complex (69.2 kDa).

Figure 3.

Nup358 binds to BicD2 with high affinity. (A) The ITC titration curve of BicD2-CTD with Nup358-RBD2 is shown, from which the affinity was determined to 0.5 ± 0.07 μM. (B) ITC titration curve of the BicD2-CTD with the Nup358-RBD2/RanGTP complex. The affinity was determined to be 3.8 ± 0.4 μM. Note that RanGTP is a negative regulator of the interaction that lowers the affinity of Nup358 toward BicD2 by a factor of 10.

Figure 4.

Affinity between Rab6^GTP and BicD2-CTD was determined by ITC as 12.6 ± 1.2 μM. The ITC titration curve of BicD2-CTD with Rab6^GTP is shown.

Figure 5.

Nup358 competes efficiently with Rab6^GTP for binding of BicD2-CTD. (A) Purified Nup358-RBD2 and BicD2-CTD were mixed in a 1:1 molar ratio and analyzed by size exclusion chromatography. An SDS-PAGE of the elution fractions is shown. Masses of molecular weight standards are shown on the left and elution volumes on the bottom. (B) The same analysis was performed for Rab6^GTP and BicD2-CTD. (C) In a competition assay, Nup358-RBD2, Rab6^GTP, and BicD2-CTD were mixed in a 1:1:1 molar ratio and the mixture was analyzed by size exclusion chromatography. (D−F) As controls, the individual proteins were analyzed: (D) Nup358-RBD2, (E) Rab6^GTP, and (F) BicD2-CTD.

Figure 6.

Proposed regulatory mechanisms for cargo selection for BicD2/dynein-dependent transport events. (A, B) BicD2 switches between recognizing vesicles and the nucleus for transport in a cell cycle-specific manner. (A) During the G1 and S phases, BicD2 mainly recognizes Rab6-positive secretory and Golgi-derived vesicles as cargo for transport. (B) The cell nucleus is recognized as cargo for dynein by BicD2 specifically in G2 phase. (C) In the G1 and S phases, BicD2 predominantly recruits dynein to Rab6-positive vesicles. Since the affinity of Nup358 toward BicD2 is high, a negative regulatory mechanism is likely required to avert BicD2 recruitment to the nucleus outside of G2 phase. A candidate is RanGTP; however, an additional negative regulator likely remains to be identified. (D) Our results suggest that the affinity of Nup358 is strong enough to efficiently compete with Rab6a^GTP for BicD2. Therefore, in the absence of negative regulation, Nup358 is expected to recruit ~1000 BicD2 molecules to the nucleus.