Neurofilament Protein Heterotetramers as Assembly Intermediates

Evidence is presented for the existence of a soluble heterotetramer containing the low and middle molecular weight neurofilament (NF) proteins, NF-L and NF-M, and one containing the low and high molecular weight proteins, NF-L and NF-H, and for their role in filament assembly. When a mixture of either pair of proteins was renatured in 2 M urea, 20 m M Tris, pH 7.2, a new band representing a complex was observed in native gel electrophoresis. No new band was observed with a mixture of NF-M and NF-H. Two-dimensional gel electrophoresis showed that treatment of the complexes with SDS caused them to dissociate into their constituent polypeptide chains. Native neurofilaments dissociated in 2 M urea into a mixture of LM and LH complexes. Titration of NF-L with NF-M indicated that complex formation was complete at an approximately equimolar ratio of the two proteins. The LM complex had a sedimentation coefficient, s20,w, of 4.4 S, consistent with a tetrameric structure. Dialysis of a solution of the LM complex against 50 m M 4-morpholineethanesulfonic acid, 0.17 M NaCl, pH 6.25, led to the formation of 10-nm filaments in good yield. These results suggest that NF protein heterooligomers are intermediates in NF assembly and disassembly. Evidence is presented for the existence of a soluble heterotetramer containing the low and middle molecular weight neurofilament (NF) proteins, NF-L and NF-M, and one containing the low and high molecular weight proteins, NF-L and NF-H, and for their role in filament assembly. When a mixture of either pair of proteins was renatured in 2 M urea, 20 m M Tris, pH 7.2, a new band representing a complex was observed in native gel electrophoresis. No new band was observed with a mixture of NF-M and NF-H. Two-dimensional gel electrophoresis showed that treatment of the complexes with SDS caused them to dissociate into their constituent polypeptide chains. Native neurofilaments dissociated in 2 M urea into a mixture of LM and LH complexes. Titration of NF-L with NF-M indicated that complex formation was complete at an approximately equimolar ratio of the two proteins. The LM complex had a sedimentation coefficient, s20,w, of 4.4 S, consistent with a tetrameric structure. Dialysis of a solution of the LM complex against 50 m M 4-morpholineethanesulfonic acid, 0.17 M NaCl, pH 6.25, led to the formation of 10-nm filaments in good yield. These results suggest that NF protein heterooligomers are intermediates in NF assembly and disassembly.