ELSEVIER Biochimica et Biophysica Acta 1253 (1995) 25-32 BB Biochi ~mic~a et Biophysica A~ta The ATPase activity of purified CDC48p from Saccharomyces cereuisiae shows complex dependence on ATP-, ADP-, and NADH-concentrations and is completely inhibited by NEM Kai-Uwe Fr6hlich a,*, Hans-Werner Fries a,b, Jan-Michael Peters c,1, Dieter Mecke a a Physiologisch-chemisches lnstitut, Hoppe-Seyler-Strafie 4, 72076 T'tibingen, Germany b Current address: lnstitutfiir medizinische Strahlenkunde und Zellforschung, Versbacher Str. 5, 97078 Wffrzburg, Germany c Deutsches Krebsforschungszentrum, Abt. fiir Zellbiologie, lm Neuenheimer Feld 280, 69120 Heidelberg, Germany Received 10 February 1995; accepted 30 May 1995 Abstract The cell cycle protein CDC48p from Saccharomyces cerecisiae is a member of a protein superfamily (AAA superfamily) characterized by a common region of approximately 200 amino-acid residues including an ATP binding consensus. CDC48p purified to homogeneity showed considerable ATPase activity which could be completely abolished by preincubation with NEM in the absence of ATP. ATP protects the protein from NEM and stabilizes the otherwise labile enzyme. The ATPase activity is reversibly inhibited by NADH and shows cooperativity with its substrate ATP. The application of the in vitro ATPase activity to the identification of physiologically interacting molecules is discussed. By electron microscopy, the enzyme was shown to consist of hexameric ring structures similar to its vertebrate homologue. Kevwords: CDC48p protein; ArPase activity; Inhibition; Cell cycle protein; N-Ethylmaleimide; (S. cereL, isiae) 1. Introduction A new protein superfamily sharing a common region of homology of approx. 200 amino-acid residues has been described recently [1-3]. Grouping according to sequence similarities and to diffelences in the composition of the sequences reveals five families within the superfamily, which appear to correspond to five different cellular func- tions. The largest group is characterized by a single copy of the common region and the absence of membrane span- ning domains. Its members, e.g., human $4 and MSSI, SUG1 protein from Saccharomyces cerecisiae, and mts2 Abbreviations: NEM, N-ethylmaleimide; NSF, NEM-sensitive fusion protein; PEP, phosphoenolpyruvate; RT, room temperature; SNAP, solu- ble NSF attachment protein. * Corresponding author. Tel.: +49 7071 293360; fax: +49 7071 296390; e-mail: kaifr@uni-tuebingen.de. i Present address: Harvard Medical School, Dept. of Cell Biology, 25 Shattuck Street, Boston, MA 0'.2115, USA. 0167-4838/95/$09.50 © 1995 Elsevier Science B.V. All rights reserved SSDI 0167-4838(95)00136-0 protein from Schizosaccharomyces pombe, appear to be subunits of the eukaryotic 26S proteasome [4-7]. Proteins containing both a membrane spanning domain and one copy of the common region have been found in eubacteria ( E. coli fish [8]) and mitochondria (S. cere- visiae YMEI [9]). ftsH is essential for cell proliferation and additionally stimulates the degradation of a regulator of )t phage lysogeny [10]. The members of the other three families contain two copies of the common region in tandem. In Secl8p from Saccharomyces cereuisiae [11] and its hamster homologue NSF [12], the second copy of the region is conserved only weakly. The proteins mediate vesicle transport from the ER through the Golgi apparatus [13] and (NSF) vesicle docking in the synaptic cleft [14]. They bind to membrane proteins (SNAREs) on both vesicles and target membranes via a set of soluble attachment proteins (a-, /3-, y-SNAP), thus initiating vesicle fusion. In a number of proteins essential for peroxisome forma- tion, the first copy of the region is conserved very weakly (e.g., S. cerevisiae Paslp [2]; Yarrowia lipolytica PAY4 protein [ 15]).