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Phospholipase D as an effector for ADP-ribosylation factor in the regulation of vesicular traffic
M G Roth1, K Bi, N T Ktistakis
1Department of Biochemistry, University of Texas Southwestern Medical Center at Dallas 75229, USA.
Abstract:
A mammalian phospholipase D (PLD) activity that is stimulated by ADP-ribosylation factor (ARF) has been identified in Golgi-enriched membrane fractions. This activity is due to the PLD1 isoform and evidence from several laboratories indicates that PLD1 is important for the polymerization of vesicle coat proteins on membranes. When expressed in Chinese hamster ovary cells, PLD1 localized to dispersed small vesicles that overlapped with the location of the ERGIC53 protein, a marker for the endoplasmic reticulum (ER)-Golgi intermediate compartment. Cells having increased PLD1 expression had accelerated anterograde and retrograde transport between the ER and Golgi. Membranes from cells having elevated PLD1 activity bound more COPI, ARF, and ARF-GTPase activating protein. These membranes also produced more COPI vesicles than did membranes from control cells. It is likely that PLD1 participates in both positive and negative feedback regulation of the formation of COPI vesicles and is important for controlling the rate of this process.
Insights
Phospholipase D1 (PLD1) regulates vesicle transport in cells. This study shows PLD1 is crucial for COPI vesicle formation and controls the rate of ER-Golgi transport.
Area of Science:
- Cell Biology
- Molecular Biology
- Biochemistry
Background:
- ADP-ribosylation factor (ARF) stimulates a mammalian phospholipase D (PLD) activity.
- PLD1 isoform is implicated in vesicle coat protein polymerization on membranes.
Purpose of the Study:
- To investigate the role of PLD1 in vesicle trafficking.
- To elucidate the mechanism by which PLD1 influences COPI vesicle formation and transport rates.
Main Methods:
- Expression of PLD1 in Chinese hamster ovary (CHO) cells.
- Localization studies using ERGIC53 as a marker.
- Analysis of anterograde and retrograde transport rates.
- Biochemical assays to measure membrane binding and vesicle production.
Main Results:
- PLD1 localized to ER-Golgi intermediate compartment vesicles.
- Increased PLD1 expression accelerated ER-Golgi transport.
- Elevated PLD1 activity enhanced binding of COPI, ARF, and ARF-GAP, increasing COPI vesicle production.
Conclusions:
- PLD1 is essential for COPI vesicle formation and regulates the rate of ER-Golgi transport.
- PLD1 likely participates in feedback regulation of COPI vesicle dynamics.