Abstract
The N-ethylmaleimide-sensitive factor (NSF), which is involved in the multisteps of protein transport, is released from Golgi membranes on in vitro incubation with Mg(2+)-ATP. However, several lines of evidence suggest that NSF is associated with membranes in spite of the presence of Mg2+ and ATP in vivo. We have used digitonin-permeabilized PC12 cells to investigate the mechanism underlying the association of NSF with membranes. In PC12 cells, immunoreactivity for NSF was observed in the nuclear membranes, the Golgi apparatus, and neuronal growth cones, where synaptic vesicles are concentrated. NSF associated with the Golgi apparatus was released on incubation with Mg(2+)-ATP, whereas NSF in the nuclear membranes and neuronal growth cones was not released on the same treatment. The addition of cytosol blocked the Mg(2+)-ATP-induced release of NSF from the Golgi apparatus. Chromatographic analyses revealed that the factor(s) that prevents NSF release from the Golgi apparatus was eluted at the same position as the soluble NSF attachment proteins (SNAPs). Purified His6-tagged alpha-SNAP exhibited such activity. His6-tagged alpha-SNAP also prevented the Mg(2+)-ATP-induced release of NSF from isolated Golgi membranes.
Highlights
The N-ethylmaleimide-sensitive factor (NSF)1 was originally characterized as a protein that is implicated in intra-Golgi vesicle-mediated protein transport [1, 2]
We examined whether Mg2ϩ-ATP induces the release of NSF from the Golgi apparatus in digitonin-permeabilized PC12 cells, as observed in isolated Golgi membranes [1]
This effect was dose-dependent, and almost complete inhibition of the release of NSF was observed at cytosol concentrations of 200 –700 g/ml. The cytosol lost this inhibitory activity on heat treatment. These results suggest the presence of a protein factor(s) in bovine brain cytosol that prevents Mg2ϩ-induced NSF release from the Golgi apparatus
Summary
Materials—PC12 cells were purchased from the Riken Cell Bank. Chinese hamster ovary Golgi membranes were prepared as described by Balch et al [19]. Monoclonal anti-NSF (2E5) was prepared as described previously [13]. Cell Permeabilization—PC12 cells were washed with ice-cold PBS twice and incubated in EDTA-ATP buffer (25 mM HEPES (pH 7.2) containing 4 mM EDTA, 1 mM ATP, 50 mM KCl, and 0.25 M sucrose) in the presence of 33– 40 M digitonin at 0 °C for 10 min. After washing with ice-cold EDTA-ATP buffer twice, the cells were incubated in EDTAATP buffer or Mg-ATP buffer (25 mM HEPES (pH 7.2) containing 5 mM MgCl2, 1 mM ATP, 50 mM KCl, and 0.25 M sucrose) at 0 °C for 30 min. NSF Release from Isolated Golgi Membranes—Isolated Golgi membranes were incubated in EDTA-ATP or Mg-ATP buffer (0.4 ml) in the presence or the absence of His6-tagged ␣-SNAP on ice. After centrifugation at 100,000 ϫ g for 30 min, the supernatant was concentrated with 6% trichloroacetic acid and 0.02% deoxycholate. The amount of NSF on blots was determined densitometrically with a Shimazu CS-9300PC scanning densitometer
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