Mapping of functional domains of gamma-SNAP.
gamma-Soluble N-ethylmaleimide-sensitive factor (NSF) attachment protein (gamma-SNAP) is capable of stabilizing a 20 S complex consisting of NSF, alpha-SNAP, and SNAP receptors (SNAREs), but its function in vesicular transport is not fully understood. Our two-hybrid analysis revealed that gamma-SNAP, unlike alpha-SNAP, interacts directly with NSF, as well as Gaf-1/Rip11, but not with SNAREs. Gaf-1/Rip11 is a gamma-SNAP-associated factor that belongs to the Rab11-interacting protein family. To gain insight into the molecular basis for the interactions of gamma-SNAP with NSF and Gaf-1/Rip11, we determined the regions of the three proteins involved in protein-protein interactions. gamma-SNAP bound to NSF via its extreme C-terminal region, and the full-length NSF was needed to interact with gamma-SNAP. Both the N-terminal and C-terminal regions of gamma-SNAP were required for the binding to Gaf-1/Rip11. Gaf-1/Rip11 bound to gamma-SNAP via its C-terminal domain comprising a putative coiled-coil region. Although the C-terminal domain of Gaf-1/Rip11 also interacts with Rab11, the binding of gamma-SNAP and Rab11 to Gaf-1/Rip11 was not mutually exclusive. Rather, Gaf-1/Rip11 was capable of serving a link between gamma-SNAP and Rab11. A complex comprising gamma-SNAP and Gaf-1/Rip11 was disassembled in a process coupled to NSF-mediated ATP hydrolysis, suggesting that the interaction between gamma-SNAP and Gaf-1/Rip11 is of functional significance.
Tani
Katsuko
K
School of Life Science, Tokyo University of Pharmacy and Life Science, Hachioji, Tokyo 192-0392, Japan.
Shibata
Mika
M
Kawase
Kazuho
K
Kawashima
Hoshiko
H
Hatsuzawa
Kiyotaka
K
Nagahama
Masami
M
Tagaya
Mitsuo
M
D048868:Adaptor Proteins, Signal Transducing; D001665:Binding Sites; D002352:Carrier Proteins / Q000737:chemistry* / Q000378:metabolism*; D015723:Gene Library; D006801:Humans; D008561:Membrane Fusion; D008565:Membrane Proteins / Q000737:chemistry* / Q000378:metabolism*; D024101:Mitochondrial Proteins; D052066:N-Ethylmaleimide-Sensitive Proteins; D010446:Peptide Fragments / Q000378:metabolism; D055503:Protein Multimerization; D050765:Qa-SNARE Proteins; D052067:Soluble N-Ethylmaleimide-Sensitive Factor Attachment Proteins; D015534:Trans-Activators / Q000378:metabolism; D020798:Two-Hybrid System Techniques; D033921:Vesicular Transport Proteins; D016335:Zinc Fingers
D048868:Adaptor Proteins, Signal Transducing; D002352:Carrier Proteins; D008565:Membrane Proteins; D024101:Mitochondrial Proteins; D010446:Peptide Fragments; D050765:Qa-SNARE Proteins; C485422:RAB11FIP5 protein, human; D052067:Soluble N-Ethylmaleimide-Sensitive Factor Attachment Proteins; D015534:Trans-Activators; D033921:Vesicular Transport Proteins; D052066:N-Ethylmaleimide-Sensitive Proteins