Plant proteases – an appetite for destruction (2005)

Proteolysis is an indispensable process in all living organisms. For most cellular proteins their existence is considerably shorter than that of the organism, and as a consequence most have a limited life span and are degraded by various proteases. Reasons for this seemingly wasteful turnover of proteins are varied. Many proteins accumulate degrees of damage via certain cellular events. Examples of such are biosynthetic errors, improper folding or thermal denaturation, as well as oxidative or other chemical damage. Such functional impairments will eventually lessen the activity of most proteins, and if left unchecked may restrict different metabolic activities and ultimately jeopardize cell integrity as a whole. In some situations, molecular chaperones can aid in repairing or refolding damaged proteins and returning them to their native state, but in many cases proteolysis is the only solution. Proteases perform a myriad of roles within a plant cell. Incorporated into the above ‘housekeeping’ duties of proteases is the recycling of essential amino acids. Recovery of such valuable nutrients via protein degradation is particularly crucial under conditions of nutrient deprivation. Proteases also play important regulatory roles throughout the cell that belie their apparent destructive nature. These regulatory roles are numerous and include maintaining stoichiometric amounts of enzyme subunits and correct apoprotein/cofactor ratios, controlling various metabolic pathways via the rapid turnover of key or rate-limiting enzymes, and influencing developmental programs by the turnover of key regulatory proteins. Many of these regulatory events are triggered by different environmental cues or by developmental signals (Vierstra 1993). The profound importance of proteases has been acknowledged recently by the awarding of last years Nobel Prize in Medicine for the discovery of the ubiquitin-mediated protein degradation system. Awarded principally for its roles in humans, the ubiquitin/26S proteasome system has tremendous importance to all eukaryotic organisms, and in plants has recently been comprehensively covered in two highly recommended reviews (see Moon et al. 2004, Smalle and Vierstra 2004). Such recognition of this obviously crucial proteolytic mechanism should not, however, overshadow the existence of other proteases nor underestimate their contribution to cellular integrity and function. To this end, the following series of minireviews recognizes several of the other important players within the proteolytic ‘symphony of destruction’ that occurs within all plant cells. These invited reviews from leading experts in the field are not intended as comprehensive treatises but rather as updates on the most currently relevant and intriguing aspects of each protease family. The aim of this editorial is to highlight some of those important features discussed in this issue and to hopefully peak the interest of readers to delve further into the subject. Whereas the enzymes involved in the ubiquitin-mediated protein degradation pathway are located in the cytosol and nucleus, other proteases are distributed in different cellular compartments. Among the various organelles, plastids are by far the most diverse and thereby one of the most intriguing. Chloroplasts in particular have an assortment of proteolytic enzymes distributed throughout, as are described in several of the reviews in this issue. Those chloroplast proteases best characterized are of bacterial ancestry. These include the Clp serine-type protease family in the stroma (Clarke et al. 2005), and the FtsH metalloproteases and Deg serine-type proteases associated to the thylakoid membrane network (Adam et al. 2005, Huesgen et al. 2005). Proteases can also be found associated to the envelope membranes, such as the Signal-peptide peptidase (SPP) that removes the transit peptide of numerous nuclear-encoded proteins translocated into chloroplasts via the Tic-Toc translocons (Richter et al. 2005). As for chloroplasts, mitochondria also possess a similar range of proteases of prokaryotic origin, including the Clp, FtsH and Lon proteases. Like their chloroplast counterparts, mitochondrial proteases are located within distinct compartments; the Clp and Lon proteases in the soluble matrix, and the FtsH proteases bound on either side of the inner membrane (Janska 2005). Vacuoles also possess a set of processing enzymes similar to SPP in chloroplasts (Yamada et al. 2005). These vacuolar-processing enzymes (VPE) are cysteine proteases responsible for the processing of vacuolar preproteins upon import into the vacuole. Different types are found in various tissues, ranging from seeds to vegetative structures. They are important in the maturation of seed storage proteins and other vacuolar proteins, as well as during different developmental changes such as senescence and the pathogen-induced hypersensitive response (Yamada et al. 2005). Many of the proteases inside plant organelles incorporate a molecular chaperone activity as an integral component of the proteolytic process. As has been well defined for their bacterial orthologs, the roles of this chaperone activity are to principally confer protein substrate specificity, and then unfold the substrate subsequent to binding. The unfolded substrate is then transferred to the adjacent proteolytic active site for complete degradation. This mode of action is reminiscent of that performed by the 26S proteasome, with separate 19S regulatory and 20S proteolytic particles (Moon et al. 2004). The partition of the two activities to distinct protein subunits occurs for the Clp protease in both chloroplasts and mitochondria (Clarke et al. 2005), while for the FtsH and Deg proteases the two are housed in separate domains within the same protein (Adam et al. 2005,Huesgen et al. 2005). For some, the binding and unfolding of the protein substrate requires energy in the form of ATP (i.e. Clp, FtsH and Lon proteases), whereas for others it does not (i.e. Deg protease). One of the most striking features for many proteases in higher plants is their copious number of paralogs. As revealed by the genomic sequencing of the model species Arabidopsis thaliana, many of the proteolytic systems throughout the plant cell are multiprotein families containing many paralogs. Chloroplasts and mitochondria are most conspicuous in this regard, as demonstrated by the Clp (Clarke et al. 2005), FtsH (Adam et al. 2005) and Deg (Huesgen et al. 2005) proteases. Attempts have been made to standardize the nomenclature of these numerous paralogs (Adam et al. 2001) but this remains an ongoing effort. This complexity in plants contrasts dramatically with the well-characterized orthologs of these protease families in non-photosynthetic organisms such as E. coli. Interestingly, the escalation in paralog number within these protease families can be seen first in photosynthetic organisms among cyanobacteria, the progenitor of plant chloroplasts. Nevertheless, the greater complexity of these paralogs in higher plants is remarkable and remains a subject of much scientific interest. One of the principle challenges arising from the existence of so many paralogs within certain protease families is how they interact with each other and with other proteins. A seminal example of this is the Clp proteins within chloroplasts. By analogy to its bacterial counterpart, the stromal Clp protease is thought to consist of a proteolytic core comprised of dual heptameric rings, flanked by adjacent hexameric rings of chaperone components. Studies to date suggest only a singular proteolytic core exists consisting of up to 10 distinct paralogs within the one 14-mer complex (Peltier et al. 2004). How this intricate proteolytic core is constructed remains unknown, as does how it interacts further with the various chaperone components to form an active protease. As evidence also exists of heterogeneous oligomeric complexes for other chloroplastic proteases (i.e. FtsH, Sakamoto et al. 2003), it is conceivable that mixed complexes are more the norm rather than the exception amongst protease families with multiple paralogs. Another of the challenging aspects of plant proteases is the identification of specific, native protein substrates and the various metabolic and regulatory processes thereby affected. As detailed in several of the following reviews, genetic studies have clearly demonstrated the overall functional importance of many of the featured proteases, particularly those located within chloroplasts. However, a dearth of information currently remains on the identity of specific protein substrates degraded by these proteases. Despite this absence, evidence is gradually gathering for native proteins degraded by certain proteases. One of the best examples is the rapid turnover of the D1 reaction centre protein of photosystem II, which appears to proceed progressively via the action of both Deg and FtsH proteases (Adam et al. 2005, Huesgen et al. 2005). Native substrates for the processing peptidases are more self-evident than those for other proteases simply by the nature of their function. The SPP enzyme in chloroplasts processes a broad range of protein precursors following import to yield the mature protein. It is also involved in the subsequent removal of cleaved transit peptides by additional stromal oligopeptidases (Richter et al. 2005). With up to 3000 nuclear-encoded proteins being transported into chloroplast, most via the Tic-Toc system, it now appears that indeed most if not all are processed by SPP. SppA peptidases also appear to be involved in the degradation of certain mature protein substrates, such as the linker proteins of the phycobilisome in some cyanobacterial strains (Sokolenko 2005). Like SPP in chloroplasts, the VPE proteases in vacuoles process a variety of imported precursor proteins in different plant tissues. VPE is particularly important in maturing seeds, responsible for the correct processing of newly arrived seed proteins within protein storage vacuoles. VPE also has a role during certain developmental transitions, such as in the disintegration of vacuolar membranes during the programmed cell death initiated by the hypersensitive response. VPE also appears to have a role in vegetative tissues at the onset of senescence and under certain stresses such as wounding (Yamada et al. 2005). Proteases are also intricately involved in developmental changes within the plant cell. As described by Rotari et al. (2005), proteases with caspase-like activity have been implicated in the programmed cell death (PCD) phenomenon in plants. These caspase-like proteases in plants are distinct from their counterparts in animals, but as yet many of their characteristics remain a mystery.