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Identification Of Cytoplasmic Residues Of Sec61p Involved In Ribosome Binding And Cotranslational Translocation, Zhiliang Cheng, Ying Jiang, Elisabet Mandon, Reid Gilmore Feb 2012

Identification Of Cytoplasmic Residues Of Sec61p Involved In Ribosome Binding And Cotranslational Translocation, Zhiliang Cheng, Ying Jiang, Elisabet Mandon, Reid Gilmore

Elisabet Mandon

The cytoplasmic surface of Sec61p is the binding site for the ribosome and has been proposed to interact with the signal recognition particle receptor during targeting of the ribosome nascent chain complex to the translocation channel. Point mutations in cytoplasmic loops six (L6) and eight (L8) of yeast Sec61p cause reductions in growth rates and defects in the translocation of nascent polypeptides that use the cotranslational translocation pathway. Sec61 heterotrimers isolated from the L8 sec61 mutants have a greatly reduced affinity for 80S ribosomes. Cytoplasmic accumulation of protein precursors demonstrates that the initial contact between the large ribosomal subunit and …


Purification Of The Golgi Adenosine 3'-Phosphate 5'-Phosphosulfate Transporter, A Homodimer Within The Membrane, Elisabet Mandon, Marcos Milla, Ellis Kempner, Carlos Hirschberg Feb 2012

Purification Of The Golgi Adenosine 3'-Phosphate 5'-Phosphosulfate Transporter, A Homodimer Within The Membrane, Elisabet Mandon, Marcos Milla, Ellis Kempner, Carlos Hirschberg

Elisabet Mandon

Sulfation of proteoglycans, secretory and membrane proteins, and glycolipids occurs in the lumen of the Golgi apparatus. Adenosine 3'-phosphate 5'-phosphosulfate (PAPS), the sulfate donor in these reactions, must be transported from the cytosol, its site of synthesis, into the lumen of the Golgi apparatus. We have identified and purified to apparent homogeneity the rat liver Golgi membrane PAPS transporter by a combination of conventional and affinity chromatography as well as photoaffinity radiolabeling with adenosine 3',5'-bisphosphate, a competitive inhibitor of PAPS transport. The transporter, a 75-kDa protein, was purified 70,000-fold over homogenate (6% yield) and transported PAPS into phosphatidylcholine liposomes selectively …