A novel assay reveals a role for soluble N-ethylmaleimide-sensitive fusion attachment protein in mannose 6-phosphate receptor transport from endosomes to the trans Golgi network.
Soluble N-ethylmaleimide-sensitive fusion protein (NSF) attachment protein (alpha-SNAP) is a soluble protein that enables the NSF ATPase to associate with membranes and facilitate membrane trafficking events. Although NSF and alpha-SNAP have been shown to be required for many membrane transport processes, their role in the transport of mannose 6-phosphate receptors from endosomes to the trans Golgi network was not established. We present here a novel in vitro assay that monitors the transport of cation-dependent mannose 6-phosphate receptors between endosomes and the trans Golgi network. The assay relies on the trans Golgi network localization of tyrosine sulfotransferase and monitors transport of mannose 6-phosphate receptors engineered to contain a consensus sequence for modification by this enzyme. Using this new assay we show that alpha-SNAP strongly stimulates transport in reactions containing limiting amounts of cytosol. Together with alpha-SNAP, NSF can increase the extent of transport. These data show that alpha-SNAP, a soluble component of the SNAP receptor machinery, facilitates transport from endosomes to the trans Golgi network.
Itin
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Department of Biochemistry, Stanford University School of Medicine, Stanford, California 94305-5307, USA.
Rancaño
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Nakajima
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Pfeffer
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D000595:Amino Acid Sequence; D000818:Animals; D001692:Biological Transport / Q000502:physiology; D016466:CHO Cells; D002352:Carrier Proteins / Q000502:physiology*; D006224:Cricetinae; D011992:Endosomes / Q000378:metabolism*; D020558:GTP Phosphohydrolases / Q000378:metabolism; D006056:Golgi Apparatus / Q000378:metabolism*; D008565:Membrane Proteins / Q000502:physiology*; D008969:Molecular Sequence Data; D017527:Receptor, IGF Type 2 / Q000378:metabolism*; D052067:Soluble N-Ethylmaleimide-Sensitive Factor Attachment Proteins; D013464:Sulfuric Acids / Q000378:metabolism; D014443:Tyrosine / Q000378:metabolism; D033921:Vesicular Transport Proteins; D020691:rab GTP-Binding Proteins
D002352:Carrier Proteins; D008565:Membrane Proteins; D017527:Receptor, IGF Type 2; D052067:Soluble N-Ethylmaleimide-Sensitive Factor Attachment Proteins; D013464:Sulfuric Acids; D033921:Vesicular Transport Proteins; D014443:Tyrosine; D020558:GTP Phosphohydrolases; D020691:rab GTP-Binding Proteins