The N-end rule pathway: from recognition by N-recognins, to destruction by AAA+proteases.

Intracellular proteolysis is a tightly regulated process responsible for the targeted removal of unwanted or damaged proteins. The non-lysosomal removal of these proteins is performed by processive enzymes, which belong to the AAA+superfamily, such as the 26S proteasome and Clp proteases. One important protein degradation pathway, that is common to both prokaryotes and eukaryotes, is the N-end rule. In this pathway, proteins bearing a destabilizing amino acid residue at their N-terminus are degraded either by the ClpAP protease in bacteria, such as Escherichia coli or by the ubiquitin proteasome system in the eukaryotic cytoplasm. A suite of enzymes and other molecular components are also required for the successful generation, recognition and delivery of N-end rule substrates to their cognate proteases. In this review we examine the similarities and differences in the N-end rule pathway of bacterial and eukaryotic systems, focusing on the molecular determinants of this pathway.

The bacterial N-end rule pathway: expect the unexpected.

The N-end rule pathway is a highly conserved process that operates in many different organisms. It relates the metabolic stability of a protein to its N-terminal amino acid. Consequently, amino acids are described as either 'stabilizing' or 'destabilizing'. Destabilizing residues are organized into three hierarchical levels: primary, secondary, and in eukaryotes - tertiary. Secondary and tertiary destabilizing residues act as signals for the post-translational modification of the target protein, ultimately resulting in the attachment of a primary destabilizing residue to the N-terminus of the protein. Regardless of their origin, proteins containing N-terminal primary destabilizing residues are recognized by a key component of the pathway. In prokaryotes, the recognition component is a specialized adaptor protein, known as ClpS, which delivers target proteins directly to the ClpAP protease for degradation. In contrast, eukaryotes use a family of E3 ligases, known as UBRs, to recognize and ubiquitylate their substrates resulting in their turnover by the 26S proteasome. While the physiological role of the N-end rule pathway is largely understood in eukaryotes, progress on the bacterial pathway has been slow. However, new interest in this area of research has invigorated several recent advances, unlocking some of the secrets of this unique proteolytic pathway in prokaryotes.

A presequence- and voltage-sensitive channel of the mitochondrial preprotein translocase formed by Tim23.

Proteins imported into the mitochondrial matrix are synthesized in the cytosol with an N-terminal presequence and are translocated through hetero-oligomeric translocase complexes of the outer and inner mitochondrial membranes. The channel across the inner membrane is formed by the presequence translocase, which consists of roughly six distinct subunits; however, it is not known which subunits actually form the channel. Here we report that purified Tim23 forms a hydrophilic, approximately 13-24 A wide channel characteristic of the mitochondrial presequence translocase. The Tim23 channel is cation selective and activated by a membrane potential and presequences. The channel is formed by the C-terminal domain of Tim23 alone, whereas the N-terminal domain is required for selectivity and a high-affinity presequence interaction. Thus, Tim23 forms a voltage-sensitive high-conductance channel with specificity for mitochondrial presequences.

The mitochondrial import machinery for preproteins.

Most mitochondrial proteins are transported from the cytosol into the organelle. Due to the division of mitochondria into an outer and inner membrane, an intermembrane space and a matrix, an elaborated system for recognition and transport of preproteins has evolved. The translocase of the outer mitochondrial membrane (TOM) and the translocases of the inner mitochondrial membrane (TIM) mediate these processes. Receptor proteins on the cytosolic face of mitochondria recognize the cargo proteins and transfer them to the general import pore (GIP) of the outer membrane. Following the passage of preproteins through the outer membrane they are transported with the aid of the TIM23 complex into either the matrix, inner membrane, or intermembrane space. Some preprotein families utilize the TIM22 complex for their insertion into the inner membrane. The identification of protein components, which are involved in these transport processes, as well as significant insights into the molecular function of some of them, has been achieved in recent years. Moreover, we are now approaching a new era in which elaborated techniques have already allowed and will enable us to gather information about the TOM and TIM complexes on an ultrastructural level.

Transport of proteins into mitochondria.

Most mitochondrial proteins are nuclear-encoded and synthesised as preproteins on polysomes in the cytosol. They must be targeted to and translocated into mitochondria. Newly synthesised preproteins interact with cytosolic factors until their recognition by receptors on the surface of mitochondria. Import into or across the outer membrane is mediated by a dynamic protein complex coined the translocase of the outer membrane (TOM). Preproteins that are imported into the matrix or inner membrane of mitochondria require the action of one of two translocation complexes of the inner membrane (TIMs). The import pathway of preproteins is predetermined by their intrinsic targeting and sorting signals. Energy input in the form of ATP and the electrical gradient across the inner membrane is required for protein translocation into mitochondria. Newly imported proteins may require molecular chaperones for their correct folding.

Multistep assembly of the protein import channel of the mitochondrial outer membrane.

Proteins targeted to mitochondria are transported into the organelle through a high molecular weight complex called the translocase of the outer mitochondrial membrane (TOM). At the core of this machinery is a multisubunit general import pore (GIP) of 400 kDa. Here we report the assembly of the yeast GIP that involves two successive intermediates of 250 kDa and 100 kDa. The precursor of the channel-lining Tom40 is first targeted to the membrane via the receptor proteins Tom20 and Tom22; it then assembles with Tom5 to form the 250 kDa intermediate exposed to the intermembrane space. The 250 kDa intermediate is followed by the formation of the 100 kDa intermediate that associates with Tom6. Maturation to the 400 kDa complex occurs by association of Tom7 and Tom22. Tom7 functions by promoting both the dissociation of the 400 kDa complex and the transition from the 100 kDa intermediate to the mature complex. These results indicate that the dynamic conversion between the 400 kDa complex and the 100 kDa late intermediate allows integration of new precursor subunits into pre-existing complexes.

Import of carrier proteins into mitochondria.

Carrier proteins located in the inner membrane of mitochondria are responsible for the exchange of metabolites between the intermembrane space and the matrix of this organelle. All members of this family are nuclear-encoded and depend on translocation machineries for their import into mitochondria. Recently many new translocation components responsible for the import of carrier proteins were identified. It is now possible to describe a detailed import pathway for this class of proteins. This review highlights the contribution made by translocation components to the process of carrier protein import into mitochondria.

Sequence analysis and heterologous expression of the groE genes from Thermoanaerobacter sp. Rt8.G4.

The groE homologous genes of the anaerobic thermophile Thermoanaerobacter sp. Rt8.G4 (TRt) have been isolated, sequenced and analysed. The TRt groES and groEL encode subunits of chaperonin 10 (Cpn10) and chaperonin 60 (Cpn60) of 94 and 541 amino acids, respectively, and are arranged in that order forming the open reading frames (ORFs) of a bicistronic operon. A controlling inverted repeat of chaperone expression (CIRCE) element lies between the consensus promoter of the operon and TRt groES. At optimum growth temperature (65 degreesC) the chaperonins of TRt are expressed, but production of Cpn60 increases significantly following temperature increases of 3-10 degreesC. Functionally intact recombinant TRt chaperonins were produced in Escherichia coli. However, owing to codon incompatibility, replacement of consecutive AGA codons in the gene encoding TRt Cpn60 was necessary for optimum expression in this heterologous host.

Purification and characterization of chaperonin 60 and chaperonin 10 from the anaerobic thermophile Thermoanaerobacter brockii.

Chaperonin 60 and chaperonin 10 (GroEL and GroES homologues, respectively) have been isolated from extracts of the anaerobic thermophile Thermoanaerobacter brockii. A simple and rapid purification for chaperonin 60 made use of hydrophobic and anion-exchange chromatographies, and could be readily scaled up; approximately 2 mg pure chaperonin 60 was obtained/g cells. In contrast with all other prokaryotic chaperonin 60 proteins that have been studied, which are tetradecamers, including those from Thermus sp., the T. brockii protein is a heptamer, and as isolated was not in association with chaperonin 10. The preparation is readily crystallized using 2-propanol or poly(ethylene glycol) with MgCl2. The N-terminal amino acid sequence of this preparation is similar to other thermophilic chaperonin 60 proteins. Chaperonin 10 was purified from the flow-through of the first hydrophobic column (which bound chaperonin 60) using a more hydrophobic adsorbent to remove contaminating proteins, followed by anion-exchange chromatography. Chaperonin 10 was obtained with a yield of approximately 10% that of chaperonin 60. The subunit molecular mass of chaperonin 10 determined by electrospray mass spectrometry is 10254 +/- 0.4 Da, which is very similar to the molecular mass of Escherichia coli GroES. Similarly, the subunit size of chaperonin 60 determined by mass spectrometry is very similar to that of GroEL, at 57949 +/- 10 Da. T. brockii chaperonin 60 has an ATPase activity that is suppressed by chaperonin 10, and the two proteins together are active in protein-folding assays. Mitochondrial malate dehydrogenase was successfully refolded at 37 degrees C after denaturation in guanidine hydrochloride, using T. brockii chaperonin 60 and chaperonin 10, or chaperonin 60 and E. coli GroES. The denatured enzyme was protected from aggregation by association with chaperonin 60. Guanidine-hydrochloride-denatured preparations of isocitrate dehydrogenase and secondary alcohol dehydrogenase isolated from T. brockii were also refolded at 60-65 degrees C. In each case, refolding required chaperonin 60, chaperonin 10 and ATP, giving up to 80% regeneration of control activity.