Roles of homooligomerization and membrane association in ATPase and proteolytic activities of FtsH in vitro.

Escherichia coli FtsH is a membrane-bound and ATP-dependent protease which degrades some soluble and integral membrane proteins. The N-terminal region of FtsH mediates membrane association as well as homooligomeric interaction of this enzyme. Previously, we studied in vivo functionality of FtsH derivatives, in which the N-terminal membrane region was either deleted (FtsH(DeltaTM)), replaced by a leucine zipper (Zip-FtsH(DeltaTM)), or replaced by a lactose permease transmembrane segment (LacY-FtsH). It was indicated that homooligomerization is required for the minimum proteolytic activity, whereas a transmembrane sequence is required for membrane protein degradation. Here we characterized these proteins in vitro. Although these mutant enzymes were very low in their activities, they were significantly stimulated by dimethyl sulfoxide, which enabled us to characterize their activities. LacY-FtsH degraded both soluble and membrane proteins, but Zip-FtsH(DeltaTM) only degraded soluble proteins. These proteins also exhibited significant ATPase activities. However, FtsH(DeltaTM) remained inactive both in ATPase and in protease activities even in the presence of dimethyl sulfoxide. The monomeric FtsH(DeltaTM) was able to bind ATP and a denatured protein. These results indicate that subunit association is important for the enzymatic catalysis by FtsH and that the additional presence of the transmembrane sequence is required for this enzyme to degrade a membrane protein even under detergent-solubilized conditions.     

Length recognition at the N-terminal tail for the initiation of FtsH-mediated proteolysis

FtsH-mediated proteolysis against membrane proteins is processive, and presumably involves dislocation of the substrate into the cytosol where the enzymatic domains of FtsH reside. To study how such a mode of proteolysis is initiated, we manipulated N-terminal cytosolic tails of three membrane proteins. YccA, a natural substrate of FtsH was found to require the N-terminal tail of 20 amino acid residues or longer to be degraded by FtsH in vivo. Three unrelated sequences of this segment conferred the FtsH sensitivity to YccA. An artificially constructed TM9-PhoA protein, derived from SecY, as well as the SecE protein, were sensitized to FtsH by addition of extra amino acid sequences to their N-terminal cytosolic tails. Thus, FtsH recognizes a cytosolic region of sufficient length (~20 amino acids) to initiate the processive proteolysis against membrane proteins. Such a region is typically at the N-terminus and can be diverse in amino acid sequences.     

Hexameric ring structure of the ATPase domain of the membrane-integrated metalloprotease FtsH from Thermus thermophilus HB8

FtsH is a cytoplasmic membrane-integrated, ATP-dependent metalloprotease, which processively degrades both cytoplasmic and membrane proteins in concert with unfolding. The FtsH protein is divided into the N-terminal transmembrane region and the larger C-terminal cytoplasmic region, which consists of an ATPase domain and a protease domain. We have determined the crystal structures of the Thermus thermophilus FtsH ATPase domain in the nucleotide-free and AMP-PNP- and ADP-bound states, in addition to the domain with the extra preceding segment. Combined with the mapping of the putative substrate binding region, these structures suggest that FtsH internally forms a hexameric ring structure, in which ATP binding could cause a conformational change to facilitate transport of substrates into the protease domain through the central pore.     

Dislocation of membrane proteins in FtsH-mediated proteolysis.

Escherichia coli FtsH degrades several integral membrane proteins, including YccA, having seven transmembrane segments, a cytosolic N-terminus and a periplasmic C-terminus. Evidence indicates that FtsH initiates proteolysis at the N-terminal cytosolic domain. SecY, having 10 transmembrane segments, is also a substrate of FtsH. We studied whether and how the FtsH-catalyzed proteolysis on the cytosolic side continues into the transmembrane and periplasmic regions using chimeric proteins, YccA-(P3)-PhoA-His6-Myc and SecY-(P5)-PhoA, with the alkaline phosphatase (PhoA) mature sequence in a periplasmic domain. The PhoA domain that was present within the fusion protein was rapidly degraded by FtsH when it lacked the DsbA-dependent folding. In contrast, both PhoA itself and the TM9-PhoA region of SecY-(P5)-PhoA were stable when expressed as independent polypeptides. In the presence of DsbA, the FtsH-dependent degradation stopped at a site near to the N-terminus of the PhoA moiety, leaving the PhoA domain (and its C-terminal region) undigested. The efficiency of this degradation stop correlated well with the rapidity of the folding of the PhoA domain. Thus, both transmembrane and periplasmic domains are degraded by the processive proteolysis by FtsH, provided they are not tightly folded. We propose that FtsH dislocates the extracytoplasmic domain of a substrate, probably using its ATPase activity.     

Topology and subcellular localization of FtsH protein in Escherichia coli.

FtsH protein in Escherichia coli is an essential protein of 70.7 kDa (644 amino acid residues) with a putative ATP-binding sequence. Western blots (immunoblots) of proteins from fractionated cell extracts and immunoelectron microscopy of the FtsH-overproducing strain showed exclusive localization of the FtsH protein in the cytoplasmic membrane. Most of the FtsH-specific labeling with gold particles was observed in the cytoplasmic membrane and the adjacent cytoplasm; much less was observed in the outer membrane and in the bulk cytoplasm. Genetic analysis by TnphoA insertions into ftsH revealed that the 25- to 95-amino-acid region, which is flanked by two hydrophobic stretchs, protrudes into the periplasmic space. From these results, we concluded that FtsH protein is an integral cytoplasmic membrane protein spanning the membrane twice and that it has a large cytoplasmic carboxy-terminal part with a putative ATP-binding domain. The average number of FtsH molecules per cell was estimated to be approximately 400.     

Membrane protein degradation by FtsH can be initiated from either end

FtsH, a membrane-bound metalloprotease, with cytoplasmic metalloprotease and AAA ATPase domains, degrades both soluble and integral membrane proteins in Escherichia coli. In this paper we investigated how membrane-embedded substrates are recognized by this enzyme. We showed previously that FtsH can initiate processive proteolysis at an N-terminal cytosolic tail of a membrane protein, by recognizing its length (more than 20 amino acid residues) but not exact sequence. Subsequent proteolysis should involve dislocation of the substrates into the cytosol. We now show that this enzyme can also initiate proteolysis at a C-terminal cytosolic tail and that the initiation efficiency depends on the length of the tail. This mode of degradation also appeared to be processive, which can be aborted by a tightly folded periplasmic domain. These results indicate that FtsH can exhibit processivity against membrane-embedded substrates in either the N-to-C or C-to-N direction. Our results also suggest that some membrane proteins receive bidirectional degradation simultaneously. These results raise intriguing questions about the molecular directionality of the dislocation and proteolysis catalyzed by FtsH.     

Molecular cloning and characterization of human kinectin.

We have identified a human cDNA that is homologous to the chicken kinectin, a putative receptor for the organelle motor kinesin. The human cDNA clone hybridized to a single 4.6-kb mRNA species that codes for a protein of 156 kDa molecular mass. The predicted primary translation product contains an N-terminal transmembrane helix followed by a bipartite nuclear localization sequence and two further C-terminal leucine zipper motifs. In addition, the aminoacid sequence revealed a large region (327-1362) of predicted alpha-helical coiled coils. A monoclonal antibody CT-1 raised against a GST-kinectin fusion protein produced a perinuclear, endoplasmic reticulum-like staining pattern in diverse cell types from different species, indicating evolutionary conservation. Monoclonal antibody CT-1 and anti-chicken kinectin antibodies cross-reacted both in Western blotting and immunoprecipitation with a 160-kDa protein, confirming the antigenic identity of this 160-kDa protein with chicken kinectin. Epitope tagging studies revealed that the nuclear localization sequence motif of kinectin is not functional. Furthermore, a truncated kinesin cDNA lacking the N-terminal hydrophobic domain revealed a nonspecific cytoplasmic staining pattern. Together the data suggest that kinectin is an integral membrane protein anchored in the endoplasmic reticulum via a transmembrane domain.     

Self-processing of FtsH and its implication for the cleavage specificity of this protease.

FtsH, a membrane-bound and ATP-dependent protease of Escherichia coli, is involved in degradation of some of uncomplexed integral membrane proteins and short-lived cytoplasmic proteins. It is composed of an N-terminal membrane-spanning region and a following large cytoplasmic domain that contains ATPase and protease active sites. In the present study, it was found that FtsH undergoes C-terminal processing in vivo. The processing was blocked by loss of function mutations of FtsH. Purified FtsH-His(6)-Myc, a C-terminally tagged derivative of FtsH, was self-processed in vitro. This in vitro processing was observed only in the presence of ATP and not in the presence of adenosine 5'-(beta,gamma-imino)triphosphate (AMP-PNP). Moreover, such processing did not occur in the case of the ATPase motif mutant protein. These results indicated that this processing is a self-catalyzed reaction that needs ATP hydrolysis. Mutations in the hflKC genes that encode a possible modulator of FtsH, and the growth phase of the cells as well, affected the processing. Complementation experiments with genetically constructed variants suggested that both the processed and the unprocessed forms of FtsH are functional. The cleavage was found to occur between Met-640 and Ser-641, removing a heptapeptide from the C-terminus of FtsH. Systematic mutational analyses of Met-640 and Ser-641 revealed preferences for positively charged and hydrophobic amino acid residues at these positions for processing. This cleavage specificity may be shared by the self-cleavage and the substrate-cleavage reactions of this protease.     

DNA-binding specificity and dimerization of the DNA-binding domain of the PEND protein in the chloroplast envelope membrane

The PEND protein is a DNA-binding protein in the inner envelope membrane of a developing chloroplast, which may anchor chloroplast nucleoids. Here we report the DNA-binding characteristics of the N-terminal basic region plus leucine zipper (bZIP)-like domain of the PEND protein that we call cbZIP domain. The basic region of the cbZIP domain diverges significantly from the basic region of known bZIP proteins that contain a bipartite nuclear localization signal. However, the cbZIP domain has the ability to dimerize in vitro. Selection of binding sites from a random sequence pool indicated that the cbZIP domain preferentially binds to a canonical sequence, TAAGAAGT. The binding site was also confirmed by gel mobility shift analysis using a representative binding site within the chloroplast DNA. These results suggest that the cbZIP domain is a unique DNA-binding domain of the chloroplast protein.     

Mutations in the leucine zipper of the human immunodeficiency virus type 1 transmembrane glycoprotein affect fusion and infectivity.

Many retroviruses, including the human and simian immunodeficiency viruses, contain a leucine zipper-like repeat in a highly conserved region of the external domain of the transmembrane (TM) glycoprotein. This region has been postulated to play a role in stabilizing the oligomeric form of these molecules. To determine what role this region might play in envelope structure and function, several mutations were engineered into the middle isoleucine of the leucine zipper-like repeat of the human immunodeficiency virus type 1 (HIV-1) TM protein. A phenotypic analysis of these mutants demonstrated that conservative mutations (Ile to Val or Leu) did not block the ability of the viral glycoprotein to mediate cell-cell fusion or affect virus infectivity. In contrast, each of the other mutations, except for the Ile-to-Ala change, completely inhibited the ability of the glycoprotein to fuse HeLa-T4 cells and of mutant virions to infect H9 cells. The alanine mutation produced an intermediate phenotype in which both cell fusion and infectivity were significantly reduced. Thus, the biological activity of the glycoprotein titrates with the hydrophobicity of the residue in this position. None of the mutations affected the synthesis, oligomer formation, transport, or processing of the HIV glycoprotein complex. Although these results do not rule out a role for the leucine zipper region in glycoprotein oligomerization, they clearly point to a critical role for it in a post-CD4 binding step in HIV membrane fusion and virus entry.     

Characterization of a conserved alpha-helical, coiled-coil motif at the C-terminal domain of the ATP-dependent FtsH (HflB) protease of Escherichia coli

FtsH (HflB) is an ATP-dependent protease found in prokaryotic cells, mitochondria and chloroplasts. Here, we have identified, in the carboxy-terminal region of FtsH (HfIB), a short alpha helix predicted of forming a coiled-coil, leucine zipper, structure. This region appears to be structurally conserved. The presence of the coiled-coil motif in the Escherichia coli FtsH (HflB) was demonstrated by circular dichroism and cross-linking experiments. Mutational analysis showed that three highly conserved leucine residues are essential for FtsH (HfIB) activity in vivo and in vitro. Purified proteins mutated in the conserved leucine residues, were found to be defective in the degradation of E. coli sigma(32) and the bacteriophage lambda CII proteins. In addition, the mutant proteins were defective in the binding of CII The mutations did not interfere with the ATPase activity of FtsH (HflB). Finally, the mutant proteins were found to be more sensitive to trypsin degradation than the wild-type enzyme suggesting that the alpha helical region is an important structural element of FtsH (HflB).     

Identification of structural and functional domains in mixed lineage kinase dual leucine zipper-bearing kinase required for complex formation and stress-activated protein kinase activation

Accumulating evidence suggests that mitogen-activated protein kinase signaling pathways form modular signaling complexes. Because the mixed lineage kinase dual leucine zipper-bearing kinase (DLK) is a large modular protein, structure-function analysis was undertaken to examine the role of DLK domains in macromolecular complex formation. DLK mutants were used to demonstrate that a DLK leucine zipper-leucine zipper interaction is necessary for DLK dimerization and to show that DLK dimerization mediated by the leucine zipper domain is prerequisite for DLK activity and subsequent activation of stress-activated protein kinase (SAPK). Heterologous mixed lineage kinase family members can be co-immunoprecipitated. However, the DLK leucine zipper domain interacted specifically only with the DLK leucine zipper domain; in contrast, DLK NH(2)-terminal region was sufficient to co-immunoprecipitate leucine zipper kinase and DLK. DLK has been shown to associate with the putative scaffold protein JIP1. This association occurred through the DLK NH(2)-terminal region and occurred independently of DLK catalytic activity. Although the DLK NH(2)-terminal region associated directly with JIP-1, this region did not interact directly with either DLK or leucine zipper kinase. Therefore, DLK may interact with heterologous mixed lineage kinase proteins via intermediary proteins. The NH(2)-terminal region of overexpressed DLK was required for activation of SAPK. These results provide evidence that protein complex formation is required for signal transduction from DLK to SAPK.     

Structure-based design of a leucine zipper protein with new DNA contacting region

We have employed a structure-based design to construct a small folding domain from the F-actin bundling protein villin that contains the amino acids necessary for the DNA binding of the basic leucine zipper protein GCN4 and have compared its DNA binding with GCN4. The monomeric motif folds into a stable domain and binds DNA in a rigid-body mechanism, while its affinity is not higher than that of the basic region peptide. The addition of the leucine zipper region to the folded domain restored its sequence-specific DNA binding comparable to that of GCN4. Unlike the monomeric folded domain, its leucine zipper derivative undergoes a conformational change upon DNA binding. CD spectral and thermodynamic studies indicate that the DNA-contacting region is folded in the presence or absence of DNA and suggest that the junction between the DNA-contacting and the leucine zipper regions transits to a helix in the presence of DNA. These results demonstrate that the structural transition outside the direct-contacting region, which adjusts the precise location of the DNA-contacting region, plays a critical role in the specific complex formation of basic leucine zipper proteins.