Related Experiment Videos
Evidence for the regulation of exocytic transport by protein phosphorylation
H W Davidson1, C H McGowan, W E Balch
1Department of Cell Biology, Scripps Research Institute, La Jolla, California 92037.
Abstract:
We investigated the effects of the protein phosphatase inhibitors okadaic acid and microcystin-LR upon transport of newly synthesized proteins through the exocytic pathway. Treatment of CHO cells with 1 microM okadaic acid rapidly inhibited movement of a marker protein (vesicular stomatitis virus G protein) from the endoplasmic reticulum to the Golgi compartment. Both okadaic acid and microcystin-LR also inhibited transport in an in vitro assay reconstituting movement to the Golgi compartment, at concentrations equivalent to those required to inhibit phosphorylase phosphatase activity. Inhibition both in vivo and in vitro could be antagonized by protein kinase inhibitors, suggesting that protein phosphorylation was directly responsible for this effect. An early stage in the transport reaction associated with vesicle formation or targeting was inhibited by protein phosphorylation, which could be reversed by fractions enriched in protein phosphatase 2A. Protein kinase antagonists did not inhibit transport between sequential compartments of the exocytic pathway in vitro, suggesting that protein phosphorylation is not itself required for vesicular transport. During mitosis, vesicular transport is inhibited simultaneous to the activation of maturation-promoting factor. It is proposed that the inhibition caused by okadaic acid and microcystin-LR involves a similar mechanism to that responsible for the mitotic arrest of vesicular transport.
Insights
Protein phosphatase inhibitors, okadaic acid and microcystin-LR, block protein transport in the exocytic pathway by promoting phosphorylation. This disruption affects vesicle formation and targeting, similar to mechanisms seen during mitotic arrest.
Area of Science:
- Cell Biology
- Molecular Biology
- Biochemistry
Background:
- The exocytic pathway is crucial for protein transport and secretion.
- Protein phosphorylation plays a role in regulating cellular processes, including protein trafficking.
Purpose of the Study:
- To investigate the impact of protein phosphatase inhibitors on protein transport via the exocytic pathway.
- To elucidate the role of protein phosphorylation in regulating vesicular transport.
Main Methods:
- Utilized Chinese Hamster Ovary (CHO) cells for in vivo studies.
- Employed an in vitro assay to reconstitute Golgi-bound transport.
- Administered okadaic acid and microcystin-LR as protein phosphatase inhibitors.
- Used protein kinase inhibitors and protein phosphatase 2A fractions for antagonism and reversal studies.
Main Results:
- Okadaic acid and microcystin-LR inhibited the movement of newly synthesized proteins from the endoplasmic reticulum to the Golgi apparatus.
- Inhibition occurred in both live cells and in vitro assays at concentrations correlating with phosphatase inhibition.
- Protein kinase inhibitors antagonized the inhibitory effects, indicating a role for protein phosphorylation.
- An early stage of transport, involving vesicle formation or targeting, was identified as the sensitive step.
Conclusions:
- Protein phosphorylation, induced by protein phosphatase inhibitors, disrupts early stages of exocytic protein transport.
- The mechanism of inhibition by okadaic acid and microcystin-LR may mirror the inhibition of vesicular transport during mitotic arrest.
- Protein phosphorylation is implicated in regulating, rather than being essential for, vesicular transport itself.