Protein unfolding in the cell Sumit Prakash and Andreas Matouschek Department of Biochemistry, Molecular Biology and Cell Biology, Northwestern University, 2153 Sheridan Road, Evanston, IL 60208, and Robert H. Lurie Comprehensive Cancer Center, Northwestern University, Chicago, IL 60611, USA Protein unfolding is an important step in several cellular processes such as protein degradation by ATP-depen- dent proteases and protein translocation across some membranes. Recent studies have shown that the mechanisms of protein unfolding in vivo differ from those of the spontaneous unfolding in vitro measured by solvent denaturation. Proteases and translocases pull at a substrate polypeptide chain and thereby catalyze unraveling by changing the unfolding pathway of that protein. The unfoldases move along the polypeptide chains of their protein substrates. The resistance of a protein to unfolding is then determined by the stability of the region of its structure that is first encountered by the unfoldase. Because unfolding is a necessary step in protein degradation and translocation, the susceptibility of a substrate protein to unfolding contributes to the specificity of these pathways. Most proteins have well-defined three-dimensional struc- tures that determine their activities in the cell; however, several cellular processes require proteins to unfold from their stable native conformations. Two examples are protein translocation across some membranes and protein degradation by ATP-dependent proteases. Both processes require the polypeptide chains to translocate through narrow channels, and the diameters of these channels are too small to accommodate folded proteins. In the past few years, it has become clear that the translocation and degradation machineries actively induce the denaturation of substrates and this unfolding activity requires energy in the form of ATP or a membrane potential. In this review, we discuss the unfolding mechanisms used by ATP-dependent proteases and the mitochondrial import machinery. These two well-studied systems form the framework of our current understanding of unfolding in the cell. Protein degradation by ATP-dependent proteases ATP-dependent proteases degrade regulatory proteins and thereby control processes such as the cell cycle, gene transcription and signal transduction. In addition, these proteases degrade misfolded or damaged polypeptides and produce many of the antigenic peptides that are displayed at the cell surface for an immune response [1,2]. In eukaryotes, this degradation activity is provided mainly by the proteasome [1]. In prokaryotes and eukaryotic organelles, similar functions are performed by analogs of the proteasome such as the ClpAP, ClpXP, HslUV, Lon and FtsH proteases [2]. The proteasome is highly selective for its substrates, despite the fact that the active sites of proteolysis themselves show very little sequence speci- ficity. Discrimination is achieved by sequestering the proteolytic sites within the structure of these proteases and by tightly controlling access to these sites. The proteasome is a large multimeric cylindrical complex that consists of two parts: a central core particle that forms the proteolytic chamber, and a regulatory particle that flanks either end of the core (Figure 1) [3,4]. The proteolytic sites in the central core are accessible only through a narrow channel that runs along the long axes of the particle [5]. The width of the degradation channel is 10–15 A ˚ at its narrowest point, which ensures that even small folded proteins cannot enter the catalytic chamber nonspecifically. The regulatory particles bind to the catalytic core at the entrance of the degradation channel and control access to the proteolytic sites [6]. The regulatory particle is composed of w17 subunits (six of which are ATPases) and is involved in substrate recog- nition [7] and unfolding [8], gating of the degradation channel [9] and translocation of the unfolded substrate to the proteolytic sites [10]. Prokaryotic ATP-dependent proteases show little, if any, sequence similarity to the eukaryotic proteasome outside the ATP-binding site but their overall structure resembles that of the proteasome (Figure 1). Substrate proteins are specifically targeted to the proteasome, most commonly via the covalent attachment of several ubiquitin moieties to lysine residues in the substrate [11]. This modification mediates the association of substrates with the protease [12]. Some proteins are targeted for degradation through adaptors that bind both the substrate and the protease simultaneously [13]. A few proteasome substrates are recognized by the protease directly through specific targeting signals that are encoded in the primary sequence of the protein [14]. The latter two recognition mechanisms are similar to the way in which most prokaryotic substrates bind their proteases [2]. Initiation of degradation Although covalent modification with ubiquitin enables a substrate protein to bind to the proteasome, effective degradation requires the presence of a second signal in the substrate: namely, an unstructured region that acts as the initiation site for degradation [15]. Degradation begins with proteolysis of this initiation site and is followed by sequential hydrolysis of the rest of the protein (Figure 2). The presence of an unstructured region is required for the degradation of tightly folded proteins, and presumably this Corresponding author: Andreas Matouschek (matouschek@northwestern.edu). Available online 30 September 2004 www.sciencedirect.com 0968-0004/$ - see front matter Q 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.tibs.2004.09.011 Review TRENDS in Biochemical Sciences Vol.29 No.11 November 2004