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Identification and partial purification of PAPS translocase
J D Ozeran1, J Westley, N B Schwartz
1Department of Pediatrics, University of Chicago, Chicago, Illinois 60637, USA.
Biochemistry
|March 26, 1996
Summary
Researchers identified the PAPS translocase, a 230 kDa protein in rat liver Golgi membranes, crucial for macromolecule sulfation. This transporter facilitates the movement of 3'-phosphoadenosine 5'-phosphosulfate (PAPS) across the Golgi membrane.
Area of Science:
- Biochemistry
- Cell Biology
- Molecular Biology
Background:
- Sulfation of macromolecules in higher organisms relies on 3'-phosphoadenosine 5'-phosphosulfate (PAPS).
- PAPS is synthesized in the cytoplasm and must be transported across the Golgi membrane for enzymatic activity.
- Previous studies kinetically characterized the PAPS translocase as an antiport transporter.
Purpose of the Study:
- To identify and physically characterize the PAPS translocase from rat liver Golgi membranes.
- To confirm the identity and properties of the PAPS translocase involved in PAPS transport.
Main Methods:
- Affinity chromatography using beta-methylene PAPS matrices.
- Protease treatment of Golgi membranes.
- Hydrodynamic analysis of membrane proteins.
- Labeling studies on intact and solubilized Golgi membranes.
- Functional assays in artificial liposomes.
Main Results:
- A 230 kDa Golgi membrane protein was isolated with PAPS translocase activity.
- This 230 kDa protein is the sole PAPS binding site accessible from the cytoplasmic face of intact Golgi.
- Protease treatment reduced the protein's size and abolished translocase activity.
- Beta-methylene PAPS binding and labeling correlated with PAPS translocase activity.
Conclusions:
- The PAPS translocase is a membrane-spanning protein of approximately 230 kDa.
- This study identifies the first member of a novel class of Golgi membrane nucleotide-metabolite transporters.
- The findings provide a molecular basis for PAPS transport essential for cellular sulfation processes.