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Published on: October 8, 2015
Identification of regulators for Ypt1 GTPase nucleotide cycling
S Jones1, C J Richardson, R J Litt
1Department of Pharmacological and Physiological Sciences, The University of Chicago, Chicago, Illinois 60637, USA.
Molecular Biology of the Cell
|October 8, 1998
Summary
Researchers identified key regulatory proteins, Ypt1p-GEF and Ypt1p-GAP, crucial for yeast secretory pathway function. These factors control Ypt1p GTPase activity, essential for vesicular transport and cellular organization.
Area of Science:
- Cell Biology
- Molecular Biology
- Protein Function
Background:
- Small GTPases of the Ypt/Rab family regulate vesicular transport.
- Nucleotide cycling (GDP/GTP-bound forms) and accessory proteins are critical for Ypt/Rab function.
- Guanine nucleotide exchange factors (GEFs) and GTPase-activating proteins (GAPs) modulate this cycling, but their roles in Ypt/Rab pathways are poorly understood.
Purpose of the Study:
- To identify and characterize factors regulating nucleotide cycling of Ypt1p, essential for early yeast secretory pathway steps.
- To investigate the physiological roles of these factors in endoplasmic reticulum (ER)-to-Golgi transport.
Main Methods:
- Identification and initial characterization of Ypt1p-GEF and Ypt1p-GAP.
- Assays to measure GEF and GAP activity, including stimulation of GDP release/GTP uptake and GTP hydrolysis.
- In vitro ER-to-Golgi transport assays using dominant-negative Ypt1p mutants and cellular fractionations.
Main Results:
- Ypt1p-GEF stimulates Ypt1p GDP release/GTP uptake >10-fold and rescues ER-to-Golgi transport inhibited by Ypt1p-D124N mutant.
- Ypt1p-GAP stimulates Ypt1p GTP hydrolysis >54-fold, shows specificity for exocytic Ypt proteins, and is independent of known Ypt GAPs (GYP7, GYP1) and vesicle fusion factors (SEC18, SEC17, SEC22).
- GEF activity localizes to ER-to-Golgi acceptor fractions, while GAP activity cofractionates with donor fractions, challenging current models.
Conclusions:
- Identification of Ypt1p-GEF and Ypt1p-GAP provides crucial insights into Ypt/Rab regulation.
- The distinct localization of GEF and GAP activities suggests a novel model for Ypt/Rab function in directional vesicular transport.
- Findings advance understanding of the molecular mechanisms governing the yeast secretory pathway.
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