Abstract
N-Ethylmaleimide-sensitive factor (NSF) has been shown to be involved in numerous intracellular membrane fusion events of both the regulated and constitutive secretory pathways. Sequence analysis indicates that the NSF subunit contains two nucleotide-binding sites, both with the classical Walker A and B motifs. In this report, we examine the nucleotide binding properties of NSF. The homotrimer contains three high affinity ATP-binding sites with Kd = 30-40 nM for ATP and Kd = 2 microM for ADP. This class of binding sites did not bind AMP, adenine, or GTP. A second class of lower affinity nucleotide binding sites with a Kd = 15-20 microM was also detected. Using various mutant forms of NSF, the high affinity nucleotide-binding sites were localized to the D2 domains and the low affinity sites were localized to the D1 domains. Functionally it is these lower affinity sites in D1 that are crucial for NSF activity. Nucleotide concentration greatly affected the ability of NSF to interact with alpha-SNAP.SNARE (soluble NSF attachment protein-SNAP receptor) complex, suggesting that only when the D1 domain ATP-binding sites are occupied does NSF bind to the alpha-SNAP.SNARE complex.
Highlights
The N-ethylmaleimide-sensitive factor (NSF)1 was originally purified based on its ability to restore intercisternal transport activity to Golgi membranes that had been previously treated with the alkylating agent N-ethylmaleimide (NEM) [1, 2]
These data were subjected to Scatchard analysis (Fig. 1, inset), and a Kd for ATP binding of 30 – 40 nM was calculated. [␣-32P]ATP and [␥32P]ATP showed similar binding characteristics, suggesting that true ATP binding was being measured in these experiments with no significant nucleotide hydrolysis
We demonstrate that NSF has two classes of ATP-binding sites that are distinct in their nucleotide binding properties and functional characteristics
Summary
ATP form, the N domains extend radially from a central core, and these domains are contracted in the ADP form [36]. The D2 domain, while apparently not required for its nucleotide binding or hydrolysis capacity, is essential to oligomerization and appears to contribute to the formation of the central core of NSF [36, 38, 40]. To further our understanding of NSF and its properties we have examined the nucleotide binding characteristics of NSF. We demonstrate that each NSF oligomer contains two distinct classes of ATP-binding sites, low and high affinity, which can be distinguished based on their contribution to NSF structure and function
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