Catalytic subunits of the multicatalytic proteinase

Abstract

The multicatalytic proteinase (MCP) has been found in a wide variety of eukaryotic cells [1,2], and a similar enzyme has been identified in archaebacteria [ 3 ] . MCP is a high molecular mass (700 kDa) non-lysosomal proteinase which may be involved in both ubiquitin-dependent and ubiquitin-independent pathways of intracellular protein degradation. The proteinase complex isolated from rat liver seems to be composed of approximately 20 different components with molecular masses ranging from 22 to 34 kDa and isoelectric points ranging from 5 to 8.5 [41. The subunits appear by electron microscopy to be arranged in the shape of a hollow cylinder. The proteinase complex can cleave peptide bonds on the carboxyl side of basic, hydrophobic and acidic amino acid residues [5], and the proteolytic activities have been referred to as trypsin-like, chymotrypsin-like and peptidyl glutamyl peptide hydrolase activities, respectively. Results of studies with a variety of inhibitors have shown that these activities are catalysed at distinct sites. It is of interest to identify which components of the complex are responsible for each of the different proteolytic activities. In this study a method has been devised for selectively labelling MCP components responsible for the trypsin-like activity. The proteinase complex was isolated from fresh rat livers using a series of chromatographic steps [4]. The method of labelling involves use of a site-specific reversible inhibitor of MCP (leupeptin) and a group-specific irreversible inhibitor (N-ethylmaleimide) . Leupeptin inhibits serine and cysteine proteinases by forming a hemiacetal or thiohemiacetal with the catalytic serine or cysteine residue, respectively [7]. Leupeptin selectively inhibits the trypsin-like activity of the MCP complex in a competitive fashion at concentrations which have no effect on the other proteolytic activities. This inhibition can be reversed either by dialysis or by the addition of sodium borohydride to reduce the reactive aldehyde group. However, the recovery of activity by either of these methods was found to be slow. NEM is also an inhibitor of the trypsinlike activity of MCP [El. It also inhibits the peptidyl glutamyl peptide hydrolase activity of MCP but has little effect on the chymotrypsin-like activity. NEM is an irreversible and specific modifier of thiol groups at neutral pH, and is available in a [14C] -labelled form. Leupeptin and NEM were used together in a differential labelling protocol where MCP was first protected by leupeptin and then modified by NEM. Both inhibitors were used at concentrations which were known to fully inhibit the trypsin-like activity. Leupeptin was removed by dialysis and then [14C]NEM was added to label sites which were previously protected by leupeptin. An experiment was also carried out in the absence of leupeptin using only labelled NEM to establish how many thiol groups react with NEM. After dialysis to remove excess labelled NEM, polypeptide components of the two labelled proteinase preparations were separated by two-dimensional SDS polyacrylamide gel electrophoresis. Labelled component (s) were identified by fluorography of the dried gel. The results of these experiments showed that up to 28 moles of [l4C1NEM were incorporated in to each mole of unprotected MCP, whereas only approximately two moles of [I4C]NEM were incorporated per mole of MCP following the differential labelling protocol. These results imply protection by leupeptin of two thiol groups per MCP molecule. Fluorography of the two different labelled MCP preparations showed that almost every component of the complex can be labelled by NEM, but that in experiments designed to label only those NEM-reactive thiol groups protected by leupeptin, only one component is strongly labelled. These labelling experiments suggest that the trypsin-like activity is located in only one of the many different components of the proteinase complex. Recent results from the labelling of catalytic sites possessing the chymotrypsin-like activity also suggest that only one component is involved [9]. These findings support the view that MCP is a complex composed of distinct components which have different functions. This work was supported by the Medical Research Council. A.J.R. is a Lister Institute Research Fellow.