Lecture 4, February 14, 2003

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Lecture 4, February 14, 2003
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Last Step of NSF Purification (MonoS-FPLC)
Block et al. (1988) PNAS 85, 7852-7856
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Averaged Negative Staining EM of NSF (Similar to
other AAA-ATPases like p97, HSP104 etc.)
Hohl et al. (1998) Mol. Cell 2, 539-548
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The Case for SNAPs (Soluble NSF Attachment
Proteins)
additional cytosolic components needed for
Intra-Golgi Transport
NSF can bind to salt-stripped Golgi only in the
presence of cytosol
-- purification of 3 cytosolic proteins a,b
and g-SNAP
a-SNAP general transport factor
b-SNAP brain specific isoform
g-SNAP precise function unknown
Clary et al. (1990) Cell 61. 709-721
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Sec17p Is the Yeast a-SNAP Homologue (Transport
Assay Dependent on a-SNAP)
Clary et al. (1990) Cell 61, 709-721
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aSNAPS Binds to K-Golgi in a Saturable Manner
Whiteheart et al. (1992) J. Biol. Chem. 267,
pp12239-12243
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aSNAP Binding to K-Golgi Is Protease Sensitive
Whiteheart et al. (1992) J. Biol. Chem. 267,
pp12239-12243
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Formation of 20S-Particles
Golgi detergent extract 35S-aSNAP /- NSF
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20S-Complexes Disassemble in the Presence of MgATP
20S Complexes /- MgATP
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Isolations of SNAP Receptors (SNAREs)
Söllner et al. (1993) Nature 362, 318-324
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Söllner et al. (1993) Nature 362, 318-324
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SNAREs Are Part of 20S Complexes
Söllner et al. (1993) Nature 362, 318-324
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20S-Complexes Disassemble in the Presence of MgATP
Bovine Brain Membrane Detergent Extract aSNAP
NSF MgATPgS or MgATP
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SNARE HYPOTHESIS
Söllner et al. (1993) Nature 362, 318-324
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Early Hypothesis on SNARE Mediated Fusion
Söllner et al. (1993) Cell 75, 409-418