Coordinated regulation of bidirectional COPI transport at the Golgi by CDC42

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Abstract

The Golgi complex has a central role in the intracellular sorting of secretory proteins1,2. Anterograde transport through the Golgi has been explained by the movement of Golgi cisternae, known as cisternal maturation3,4,5. Because this explanation is now appreciated to be incomplete6, interest has developed in understanding tubules that connect the Golgi cisternae7,8,9. Here we show that the coat protein I (COPI) complex sorts anterograde cargoes into these tubules in human cells. Moreover, the small GTPase CDC42 regulates bidirectional Golgi transport by targeting the dual functions of COPI in cargo sorting and carrier formation. CDC42 also directly imparts membrane curvature to promote COPI tubule formation. Our findings further reveal that COPI tubular transport complements cisternal maturation in explaining how anterograde Golgi transport is achieved, and that bidirectional COPI transport is modulated by environmental cues through CDC42.

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Figure 1: Coatomer binds VSVG to promote its transport through the Golgi.
Figure 2: Coatomer also binds to the LDLR tail to promote the transport of VSVG–LDLR through the Golgi.
Figure 3: CDC42 modulates cargo sorting by COPI.
Figure 4: CDC42 modulates carrier formation by COPI.

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Acknowledgements

We thank J. Li, M. Bai, X. Michelet and C. Alves for discussions, and M. Ericsson for electron microscopy technical advice. This work was funded by grants from the National Institutes of Health to V.W.H. (R01GM058615), R.J.S. (R01AI068871, R01AR065538 and 1S10RR027931-01), A.B.S. (K01DK089145), and also by the Basic Science Research Program of the National Research Foundation of Korea to S.-Y.P. (2014R1A6A3A03056673).

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Contributions

S.-Y.P., J.-S.Y. and A.B.S. performed the experiments. V.W.H., S.-Y.P., R.J.S. and A.B.S. designed the experiments and wrote the paper.

Corresponding author

Correspondence to Victor W. Hsu.

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The authors declare no competing financial interests.

Extended data figures and tables

Extended Data Figure 1 Characterizing VSVG transport.

a, Cytoplasmic sequence of VSVG with residues crucial for binding by coatomer highlighted. b, Effect of point mutations in the VSVG tail on the in vitro binding of this tail by coatomer, n = 5. c, FLIM assessing interaction between VSVG and coatomer. Representative images that are pseudo-coloured based on τ1 values are shown (out of 10). Quantification is also shown, n = 3. d, Effect of mutations in the VSVG tail on its transport from the ER to the trans-Golgi. Colocalization is exemplified by line scanning across representative images (out of 5). e, Effect of mutations in the VSVG tail on its transport from the ER to the cis-Golgi. Representative images of colocalization are shown (out of 5). Quantification is also shown, n = 3. Data are mean ± s.e.m. Scale bars, 5 μm.

Extended Data Figure 2 Characterizing VSVG–LDLR transport.

a, Cytoplasmic sequence of LDLR with basic residues critical for binding by coatomer highlighted. b, Effect of mutations in the LDLR tail on the transport of VSVG–LDLR from the ER to the trans-Golgi. Colocalization is exemplified by line scanning across representative images (out of 5). c, Effect of mutations in the LDLR tail on the transport of VSVG–LDLR from the ER to the cis-Golgi. Representative images of colocalization are shown (out of 5). Quantification is also shown, n = 4, mean ± s.e.m. d, Effect of mutations in cargo tails on the intra-Golgi transport of various cargoes. Colocalization is exemplified by line scanning across representative images (out of 5). Scale bars, 5 μm.

Extended Data Figure 3 Further characterizing cargo transport.

a, FLIM assessing interaction between different forms of VSVG and coatomer. Representative images that are pseudo-coloured based on τ1 values are shown (out of 10). Quantification is also shown, n = 3. b, Effect of mutations in the VSVG tail on its transport from the ER to the trans-Golgi. Colocalization is exemplified by line scanning across representative images (out of 5). Quantification is also shown, n = 3. Data are mean ± s.e.m. Scale bars, 5 μm.

Extended Data Figure 4 Characterizing how CDC42 affects VSVG transport.

a, Effect of expressing active CDC42 on the intra-Golgi transport of VSVG. Colocalization is exemplified by line scanning across representative images (out of 5). b, Immunoblotting of whole-cell lysates to assess efficiency of siRNA treatment, n = 3. c, Effect of siRNA against CDC42 on the intra-Golgi transport of VSVG. Colocalization is exemplified by line scanning across representative images (out of 5). Quantification is also shown, n = 4. d, Effect of expressing active CDC42 on VSVG transport from the ER to the cis-Golgi. Representative images of colocalization are shown (out of 5). Quantification is also shown, n = 5. e, Effect of siRNA against CDC42 on VSVG transport from the ER to the cis-Golgi. Representative images of colocalization are shown (out of 5). Quantification is also shown, n = 4. Data are mean ± s.e.m. Scale bars, 5 μm.

Extended Data Figure 5 Further characterizing the effects of CDC42.

a, Effect of various conditions on the intra-Golgi transport of VSVG. Colocalization is exemplified by line scanning across representative images (out of 5). b, Effect of expressing active CDC42 on the transport of VSVG–KDELR. Representative images of colocalization is shown (out of 5). c, Effect of siRNA against CDC42 on the transport of VSVG–KDELR. Representative images of colocalization are shown (out of 5). Quantification is also shown, n = 3. d, e, FLIM assessing the effect of active CDC42 on the interaction between different VSVG forms and coatomer. Representative images that are pseudo-coloured based on τ1 values are shown (out of 10). Quantification is also shown as table and as graph, n = 3. ***P < 0.001 (two-tailed Mann–Whitney test). Data are mean ± s.e.m. Scale bars, 5 μm.

Extended Data Figure 6 Characterizing how CDC42 affects COPI cargo sorting and carrier formation.

a, b, Effect of different small GTPases on the in vitro binding of cargo tails by coatomer. Representative blots are shown (out of 3). Quantification is also shown, n = 3, mean ± s.e.m. *P < 0.05 (two-tailed Student’s t-test). c, d, Effect of different active forms of CDC42 on tubule and vesicle formation in the COPI reconstitution system, n = 4, mean ± s.e.m. **P < 0.01 (two-tailed Student’s t-test).

Extended Data Figure 7 Delineating the role of di-arginine residues in CDC42.

a, Amino acid sequence at the C terminus of CDC42. The last three residues are cleaved after prenylation. b, The di-arginine residues in CDC42 are required for dimerization. Representative result from gel filtration (out of 2) is shown. c, Effect of different conditions on the intra-Golgi transport of VSVG. Colocalization is exemplified by line scanning across representative images (out of 5). Scale bar, 5 μm. d, Effect of mutations in CDC42 on its ability to bind coatomer in vitro. Representative blot (out of 2) is shown. e, Effect of mutations in CDC42 on its ability to compete with cargo tails for binding to coatomer in vitro. Representative blot (out of 3) is shown. f, Effect of different forms of CDC42 to compete with the KDELR tail for binding to coatomer in vitro. Representative blot (out of 2) is shown. g, FLIM examining VSVG–KDELR interacting with coatomer, n = 3. Data are mean ± s.e.m. ***P < 0.001 (two-tailed Mann–Whitney test).

Extended Data Figure 8 Delineating how external stimuli regulate bidirectional Golgi transport.

a, Effect of different conditions on the intra-Golgi transport of VSVG. Colocalization is exemplified by line scanning across representative images (out of 5). Scale bar, 5 μm. Quantification is also shown, n = 3. b, Effect of serum on the transport of VSVG–KDELR, n = 3. c, Effect of EGF on the intra-Golgi transport of VSVG, n = 3. d, Effect of EGF on the transport of VSVG–KDELR, n = 3. e, f, Effect of different conditions on VSVG transport from the ER to the cis-Golgi, n = 3. Data are mean ± s.e.m.

Extended Data Figure 9 Delineating how SRC regulates bidirectional Golgi transport.

a, c, Effect of different conditions on the intra-Golgi transport of VSVG. Colocalization is exemplified by line scanning across representative images (out of 5). Scale bar, 5 μm. Quantification is also shown, n = 3 experiments. b, d, Effect of different conditions on the transport of VSVG–KDELR, n = 3. Data are mean ± s.e.m.

Extended Data Figure 10 Further characterizing how bidirectional COPI transport at the Golgi is regulated.

a, Summarizing how external stimuli regulates bidirectional COPI transport through a signalling cascade. b, Effect of SRC activation on the intra-Golgi transport of VSVG. Colocalization is exemplified by line scanning across representative images (out of 5). Scale bar, 5 μm. Quantification is also shown, n = 3. Data are mean ± s.e.m.

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Park, S., Yang, J., Schmider, A. et al. Coordinated regulation of bidirectional COPI transport at the Golgi by CDC42. Nature 521, 529–532 (2015). https://doi.org/10.1038/nature14457

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