J-domain protein CDJ2 and HSP70B are a plastidic chaperone pair that interacts with vesicle-inducing protein in plastids 1

J-domain cochaperones confer functional specificity to their heat shock protein (HSP)70 partner by recruiting it to specific substrate proteins. To gain insight into the functions of plastidic HSP70s, we searched in Chlamydomonas databases for expressed sequence tags that potentially encode chloroplast-targeted J-domain cochaperones. Two such cDNAs were found: the encoded J-domain proteins were named chloroplast DnaJ homolog 1 and 2 (CDJ1 and CDJ2). CDJ2 was shown to interact with a approximately 28-kDa protein that by mass spectrometry was identified as the vesicle-inducing protein in plastids 1 (VIPP1). In fractionation experiments, CDJ2 was detected almost exclusively in the stroma, whereas VIPP1 was found in low-density membranes, thylakoids, and in the stroma. Coimmunoprecipitation and mass spectrometry analyses identified stromal HSP70B as the major protein interacting with soluble VIPP1, and, as confirmed by cross-linking data, as chaperone partner of CDJ2. In blue native-PAGE of soluble cell extracts, CDJ2 and VIPP1 comigrated in complexes of >669, approximately 150, and perhaps approximately 300 kDa. Our data suggest that CDJ2, presumably via coiled-coil interactions, binds to VIPP1 and presents it to HSP70B in the ATP state. Our findings and the previously reported requirement of VIPP1 for the biogenesis of thylakoid membranes point to a role for the HSP70B/CDJ2 chaperone pair in this process.     

The C-terminal helices of heat shock protein 70 are essential for J-domain binding and ATPase activation

The J-domain co-chaperones work together with the heat shock protein 70 (HSP70) chaperone to regulate many cellular events, but the mechanism underlying the J-domain-mediated HSP70 function remains elusive. We studied the interaction between human-inducible HSP70 and Homo sapiens J-domain protein (HSJ1a), a J domain and UIM motif-containing co-chaperone. The J domain of HSJ1a shares a conserved structure with other J domains from both eukaryotic and prokaryotic species, and it mediates the interaction with and the ATPase cycle of HSP70. Our in vitro study corroborates that the N terminus of HSP70 including the ATPase domain and the substrate-binding β-subdomain is not sufficient to bind with the J domain of HSJ1a. The C-terminal helical α-subdomain of HSP70, which was considered to function as a lid of the substrate-binding domain, is crucial for binding with the J domain of HSJ1a and stimulating the ATPase activity of HSP70. These fluctuating helices are likely to contribute to a proper conformation of HSP70 for J-domain binding other than directly bind with the J domain. Our findings provide an alternative mechanism of allosteric activation for functional regulation of HSP70 by its J-domain co-chaperones.     

Mutational analysis of the human HSP70 protein: distinct domains for nucleolar localization and adenosine triphosphate binding

The human HSP70 gene was modified in vitro using oligonucleotide-directed mutagenesis to add sequences encoding a peptide from the testis-specific form of human lactate dehydrogenase (LDH) to the carboxy terminus of HSP70. The peptide-tagged HSP70 can be distinguished from the endogenous HSP70 protein using an LDH peptide-specific antiserum in indirect immunofluorescence assays of cells transiently transfected with an expression vector containing the tagged HSP70 gene regulated by the human HSP70 promoter. A series of deletion mutants within the HSP70 protein coding region were generated. Using double-label indirect immunofluorescence with the LDH peptide-specific antiserum and HSP70-specific mAbs, we compared the intracellular distribution of the deletion mutants to that of endogenous HSP70. We have determined that sequences in the carboxy terminus of HSP70 are necessary for proper nucleolar localization after heat shock. In contrast, sequences in the amino terminus of HSP70 are responsible for the ATP-binding ability of the protein. Mutants that were unable to bind ATP, however, still displayed nucleolar association, indicating that ATP binding is apparently not required for interaction with substrate. Additional support that HSP70 appears to be composed of at least two domains follows from the results of trypsin digestions of wild type and mutant HSP70. Protease digestion of the mutant HSP70 proteins identified a region of HSP70 that, when deleted, affected HSP70 conformation.     

Trafficking and assembly of the cytoadherence complex in Plasmodium falciparum-infected human erythrocytes

After invading human erythrocytes, the malarial parasite Plasmodium falciparum, initiates a remarkable process of secreting proteins into the surrounding erythrocyte cytoplasm and plasma membrane. One of these exported proteins, the knob-associated histidine-rich protein (KAHRP), is essential for microvascular sequestration, a strategy whereby infected red cells adhere via knob structures to capillary walls and thus avoid being eliminated by the spleen. This cytoadherence is an important factor in many of the deaths caused by malaria. Green fluorescent protein fusions and fluorescence recovery after photobleaching were used to follow the pathway of KAHRP deployment from the parasite endomembrane system into an intermediate depot between parasite and host, then onwards to the erythrocyte cytoplasm and eventually into knobs. Sequence elements essential to individual steps in the pathway are defined and we show that parasite-derived structures, known as Maurer's clefts, are an elaboration of the canonical secretory pathway that is transposed outside the parasite into the host cell, the first example of its kind in eukaryotic biology.     

Knobs, knob proteins and cytoadherence in falciparum malaria.

1. The sequestration of trophozoite and schizont infected erythrocytes (IRBC) in post-capillary venules of host internal organs causes most of the morbidity and mortality in falciparum malaria. It is a knob mediated cytoadherence phenomenon where knobs act as the focal junction between IRBC and host endothelial cell. Knobless (K-) parasites, isolated from cultures (not yet isolated from in vivo), do not cause virulent infections. Knobs thus play an important role in pathophysiology of falciparum malaria. 2. The chemical composition of knobs is partly explored, several proteins (Known as knob proteins) have been identified. According to their function they can be classified as (a) knob-inducing protein, "KAHRP" (b) knob-associated cytoadherent proteins, e.g. PFEMP-1, modified band 3 and an antigen recognized by monoclonal 33G2 and (c) knob-associated structural protein, e.g. PFEMP-2/MESA/PP-300. Most of them show size polymorphism among different isolates. Only KAHRP and MESA/PFEMP-2 have been studied at molecular level. Their chromosomal locations have been identified such as KAHRP on chromosome 2 and MESA/PFEMP-2 on chromosomes 5 and 6. 3. The receptor molecules on endothelial cells for knob ligands have been identified and partially characterized. 4. Knob ligands and their receptor molecules can play an important role in developing the immunotherapeutic reagents. 5. Based on the available data a tentative hypothesis has been proposed about the loss of knobs in vitro. Nevertheless, this needs further support from other experimental evidence. 6. Future work should be directed towards the structure and function of knob proteins and their interactions with each other as well as with host proteins. Regulation of expression of knobs and knob protein(s), evaluation of knob antigens for immunotherapy of severe falciparum malaria and for a malaria vaccine also require further investigations.     

Co-chaperone HSJ1a dually regulates the proteasomal degradation of ataxin-3

Homo sapiens J domain protein (HSJ1) is a J-domain containing co-chaperone that is known to stimulate ATPase activity of HSP70 chaperone, while it also harbors two ubiquitin (Ub)-interacting motifs (UIMs) that may bind with ubiquitinated substrates and potentially function in protein degradation. We studied the effects of HSJ1a on the protein levels of both normal and the disease--related polyQ-expanded forms of ataxin-3 (Atx3) in cells. The results demonstrate that the N-terminal J-domain and the C-terminal UIM domain of HSJ1a exert opposite functions in regulating the protein level of cellular overexpressed Atx3. This dual regulation is dependent on the binding of the J-domain with HSP70, and the UIM domain with polyUb chains. The J-domain down-regulates the protein level of Atx3 through HSP70 mediated proteasomal degradation, while the UIM domain may alleviate this process via maintaining the ubiquitinated Atx3. We propose that co-chaperone HSJ1a orchestrates the balance of substrates in stressed cells in a Yin-Yang manner.     

Chaperones in cell cycle regulation and mitogenic signal transduction: a review

Chaperones/heat shock proteins (HSPs) of the HSP90 and HSP70 families show elevated levels in proliferating mammalian cells and a cell cycle-dependent expression. They transiently associate with key molecules of the cell cycle control system such as Cdk4, Wee-1, pRb, p53, p27/Kip1 and are involved in the nuclear localization of regulatory proteins. They also associate with viral oncoproteins such as SV40 super T, large T and small t antigen, polyoma large and middle S antigen and EpsteinBarr virus nuclear antigen. This association is based on a J-domain in the viral proteins and may assist their targeting to the pRb/E2F complex. Small HSPs and their state of phosphorylation and oligomerization also seem to be involved in proliferation and differentiation. Chaperones/HSPs thus play important roles within cell cycle processes. Their exact functioning, however, is still a matter of discussion. HSP90 in particular, but also HSP70 and other chaperones associate with proteins of the mitogen-activated signal cascade, particularly with the Src kinase, with tyrosine receptor kinases, with Raf and the MAP-kinase activating kinase (MEK). This apparently serves the folding and translocation of these proteins, but possibly also the formation of large immobilized complexes of signal transducing molecules (scaffolding function).     

The chloroplast DnaJ homolog CDJ1 of Chlamydomonas reinhardtii is part of a multichaperone complex containing HSP70B, CGE1, and HSP90C

We report on the molecular and biochemical characterization of CDJ1, one of three zinc-finger-containing J-domain proteins encoded by the Chlamydomonas reinhardtii genome. Fractionation experiments indicate that CDJ1 is a plastidic protein. In the chloroplast, CDJ1 was localized to the soluble stroma fraction, but also to thylakoids and to low density membranes. Although the CDJ1 gene was strongly heat shock inducible, CDJ1 protein levels increased only slightly during heat shock. Cellular CDJ1 concentrations were close to those of heat shock protein 70B (HSP70B), the major HSP70 in the Chlamydomonas chloroplast. CDJ1 complemented the temperature-sensitive phenotype of an Escherichia coli mutant lacking its dnaJ gene and interacted with E. coli DnaK, hence classifying it as a bona fide DnaJ protein. In soluble cell extracts, CDJ1 was found to organize into stable dimers and into complexes of high molecular mass. Immunoprecipitation experiments revealed that CDJ1 forms common complexes with plastidic HSP90C, HSP70B, and CGE1. In blue native-polyacrylamide gel electrophoresis, all four (co)chaperones migrated at 40% to 90% higher apparent than calculated molecular masses, indicating that greatest care must be taken when molecular masses of protein complexes are estimated from their migration relative to standard native marker proteins. Immunoprecipitation experiments from size-fractioned soluble cell extracts suggested that HSP90C and HSP70B exist as preformed complex that is joined by CDJ1. In summary, CDJ1 and CGE1 are novel cohort proteins of the chloroplast HSP90-HSP70 multichaperone complex. As HSP70B, CDJ1, and CGE1 are derived from the endosymbiont, whereas HSP90C is of eukaryotic origin, we observe in the chloroplast the interaction of two chaperone systems of distinct evolutionary origin.     

CUEDC2 interacts with heat shock protein 70 and negatively regulates its chaperone activity

Recently studies have revealed that CUEDC2, a CUE domain-containing protein, plays critical roles in many biological processes, such as cell cycle, inflammation and tumorigenesis. In this study, to further explore the function of CUEDC2, we performed affinity purification combined with mass spectrometry analysis to identify its interaction proteins, which led to the identification of heat shock protein 70 (HSP70). We confirmed the interaction between CUEDC2 and HSP70 in vivo by co-immunoprecipitation assays. Mapping experiments revealed that CUE domain was required for their binding, while the PBD and CT domains of HSP70, mediated the interaction with CUEDC2. The intracellular Luciferase refolding assay indicated that CUEDC2 could inhibit the chaperone activity of HSP70. Together, our results identify HSP70 as a novel CUEDC2 interaction protein and suggest that CUEDC2 might play important roles in regulating HSP70 mediated stress responses.     

HSP70 and Its Cochaperone CPIP Promote Potyvirus Infection in Nicotiana benthamiana by Regulating Viral Coat Protein Functions

This study demonstrates that heat shock protein 70 (HSP70) together with its cochaperone CPIP regulates the function of a potyviral coat protein (CP), which in turn can interfere with viral gene expression. HSP70 was copurified as a component of a membrane-associated viral ribonucleoprotein complex from Potato virus A–infected plants. Downregulation of HSP70 caused a CP-mediated defect associated with replication. When PVA CP was expressed in trans, it interfered with viral gene expression and replication-associated translation (RAT). However, CP produced in cis interfered specifically with RAT. CPIP binds to potyviral CP, and overexpression of CPIP was sufficient to restore RAT inhibited by expression of CP in trans. Restoration of RAT was dependent on the ability of CPIP to interact with HSP70 since expression of a J-domain mutant, CPIP^Δ66, had only a minor effect on RAT. CPIP-mediated delivery of CP to HSP70 promoted CP degradation by increasing its ubiquitination when assayed in the absence of virus infection. In conclusion, CPIP and HSP70 are crucial components of a distinct translation activity that is associated with potyvirus replication.     

The Malarial Exported PFA0660w Is an Hsp40 Co-Chaperone of PfHsp70-x

Plasmodium falciparum, the human pathogen responsible for the most dangerous malaria infection, survives and develops in mature erythrocytes through the export of proteins needed for remodelling of the host cell. Molecular chaperones of the heat shock protein (Hsp) family are prominent members of the exportome, including a number of Hsp40s and a Hsp70. PFA0660w, a type II Hsp40, has been shown to be exported and possibly form a complex with PfHsp70-x in the infected erythrocyte cytosol. However, the chaperone properties of PFA0660w and its interaction with human and parasite Hsp70s are yet to be investigated. Recombinant PFA0660w was found to exist as a monomer in solution, and was able to significantly stimulate the ATPase activity of PfHsp70-x but not that of a second plasmodial Hsp70 (PfHsp70-1) or a human Hsp70 (HSPA1A), indicating a potential specific functional partnership with PfHsp70-x. Protein binding studies in the presence and absence of ATP suggested that the interaction of PFA0660w with PfHsp70-x most likely represented a co-chaperone/chaperone interaction. Also, PFA0660w alone produced a concentration-dependent suppression of rhodanese aggregation, demonstrating its chaperone properties. Overall, we have provided the first biochemical evidence for the possible role of PFA0660w as a chaperone and as co-chaperone of PfHsp70-x. We propose that these chaperones boost the chaperone power of the infected erythrocyte, enabling successful protein trafficking and folding, and thereby making a fundamental contribution to the pathology of malaria.     

An erythrocyte cytoskeleton-binding motif in exported Plasmodium falciparum proteins

Binding of exported malaria parasite proteins to the host cell membrane and cytoskeleton contributes to the morphological, functional, and antigenic changes seen in Plasmodium falciparum-infected erythrocytes. One such exported protein that targets the erythrocyte cytoskeleton is the mature parasite-infected erythrocyte surface antigen (MESA), which interacts with the N-terminal 30-kDa domain of protein 4.1R via a 19-residue sequence. We report here that the MESA erythrocyte cytoskeleton-binding (MEC) domain is present in at least 13 other P. falciparum proteins predicted to be exported to the host cell. An alignment of the putative cytoskeleton-binding sequences revealed a conserved aspartic acid at the C terminus that was omitted from the originally reported binding domain. Mutagenesis experiments demonstrated that this aspartic acid was required for the optimal binding of MESA to inside-out vesicles (IOVs) prepared from erythrocytes. Using pulldown assays, we characterized the binding of fragments encoding the MEC domains from PFE0040c/MESA and six other proteins (PF10_0378, PFA0675w, PFB0925w, PFD0095c, PFF1510w, and PFI1790w) to IOVs. All seven proteins bound to IOVs, with MESA showing the strongest affinity in saturation binding experiments. We further examined the interaction of the MEC domain proteins with components of the erythrocyte cytoskeleton and showed that MESA, PF10_0378, and PFA0675w coprecipitated full-length 4.1R from lysates prepared from IOVs. These data demonstrated that the MEC motif is present and functional in at least six other P. falciparum proteins that are exported to the host cell cytoplasm.     

A Single Protein S-acyl Transferase Acts through Diverse Substrates to Determine Cryptococcal Morphology,Stress Tolerance,and Pathogenic Outcome

Cryptococcus neoformans is an opportunistic yeast that kills over 625,000 people yearly through lethal meningitis. Host phagocytes serve as the first line of defense against this pathogen, but fungal engulfment and subsequent intracellular proliferation also correlate with poor patient outcome. Defining the interactions of this facultative intracellular pathogen with host phagocytes is key to understanding the latter’s opposing roles in infection and how they contribute to fungal latency, dissemination, and virulence. We used high-content imaging and a human monocytic cell line to screen 1,201 fungal mutants for strains with altered host interactions and identified multiple genes that influence fungal adherence and phagocytosis. One of these genes was PFA4, which encodes a protein S-acyl transferase (PAT), one of a family of DHHC domain-containing proteins that catalyzes lipid modification of proteins. Deletion of PFA4 caused dramatic defects in cryptococcal morphology, stress tolerance, and virulence. Bioorthogonal palmitoylome-profiling identified Pfa4-specific protein substrates involved in cell wall synthesis, signal transduction, and membrane trafficking responsible for these phenotypic alterations. We demonstrate that a single PAT is responsible for the modification of a subset of proteins that are critical in cryptococcal pathogenesis. Since several of these palmitoylated substrates are conserved in other pathogenic fungi, protein palmitoylation represents a potential avenue for new antifungal therapeutics.     

Identification of a Plasmodium falciparum Phospholipid Transfer Protein

Infection of erythrocytes by the human malaria parasite Plasmodium falciparum results in dramatic modifications to the host cell, including changes to its antigenic and transport properties and the de novo formation of membranous compartments within the erythrocyte cytosol. These parasite-induced structures are implicated in the transport of nutrients, metabolic products, and parasite proteins, as well as in parasite virulence. However, very few of the parasite effector proteins that underlie remodeling of the host erythrocyte are functionally characterized. Using bioinformatic examination and modeling, we have found that the exported P. falciparum protein PFA0210c belongs to the START domain family, members of which mediate transfer of phospholipids, ceramide, or fatty acids between membranes. In vitro phospholipid transfer assays using recombinant PFA0210 confirmed that it can transfer phosphatidylcholine, phosphatidylinositol, phosphatidylethanolamine, and sphingomyelin between phospholipid vesicles. Furthermore, assays using HL60 cells containing radiolabeled phospholipids indicated that orthologs of PFA0210c can also transfer phosphatidylcholine, phosphatidylinositol, and phosphatidylethanolamine. Biochemical and immunochemical analysis showed that PFA0210c associates with membranes in infected erythrocytes at mature stages of intracellular parasite growth. Localization studies in live parasites revealed that the protein is present in the parasitophorous vacuole during growth and is later recruited to organelles in the parasite. Together these data suggest that PFA0210c plays a role in the formation of the membranous structures and nutrient phospholipid transfer in the malaria-parasitized erythrocyte.     

Two-dimensional blue native/SDS-PAGE analysis reveals heat shock protein chaperone machinery involved in hepatitis B virus production in HepG2.2.15 cells

Hepatitis B virus (HBV) infection is a major health concern with more than two billion individuals currently infected worldwide. Despite the prevalence of infection, gaining a complete understanding of the molecular mechanisms of HBV infection has been difficult because HBV cannot infect common immortalized cell lines. HepG2.2.15, however, is a well established version of the HepG2 cell line that constitutively expresses HBV. Therefore, comparative proteomics analysis of HepG2.2.15 and HepG2 may provide valuable clues for understanding the HBV virus life cycle. In this study, two-dimensional blue native/SDS-PAGE was utilized to characterize different multiprotein complexes from whole cell lysates between HepG2.2.15 and HepG2. These results demonstrate that two unique protein complexes existed in HepG2.2.15 cells. When these complexes were excised from the gel and subjected to the second dimension separation and the proteins were sequenced by mass spectrometry, 20 non-redundant proteins were identified. Of these proteins, almost 20% corresponded to heat shock proteins, including HSP60, HSP70, and HSP90. Antibody-based supershift assays were used to verify the validity of the distinct protein complexes. Co-immunoprecipitation assays confirmed that HSP60, HSP70, and HSP90 proteins physically interacted in HepG2.2.15 but not HepG2 cells. We further demonstrated that down-regulation of HSP70 or HSP90 by small interfering RNA significantly inhibited HBV viral production but did not influence cellular proliferation or apoptosis. Consistent with these results, a significant reduction in HepG2.2.15 HBV secretion was observed when the HSP90 inhibitor 17-allylamino-17-demethoxygeldanamycin was used to treat HepG2.2.15 cells. Collectively these results suggest that the interaction of HSP90 with HSP70/HSP60 contributes to the HBV life cycle by forming a multichaperone machine that may constitute therapeutic targets for HBV-associated diseases.     

Crystal structure of Hsc20, a J-type Co-chaperone from Escherichia coli

Hsc20 is a 20 kDa J-protein that regulates the ATPase activity and peptide-binding specificity of Hsc66, an hsp70-class molecular chaperone. We report herein the crystal structure of Hsc20 from Escherichia coli determined to a resolution of 1.8 A using a combination of single isomorphous replacement (SIR) and multi-wavelength anomalous diffraction (MAD). The overall structure of Hsc20 consists of two distinct domains, an N-terminal J-domain containing residues 1-75 connected by a short loop to a C-terminal domain containing residues 84-171. The structure of the J-domain, involved in interactions with Hsc66, resembles the alpha-topology of J-domain fragments of Escherichia coli DnaJ and human Hdj1 previously determined by solution NMR methods. The C-terminal domain, implicated in binding and targeting proteins to Hsc66, consists of a three-helix bundle in which two helices comprise an anti-parallel coiled-coil. The two domains make contact through an extensive hydrophobic interface ( approximately 650 A(2)) suggesting that their relative orientations are fixed. Thus, Hsc20, in addition to its role in the regulation of the ATPase activity of Hsc66, may also function as a rigid scaffold to facilitate positioning of the protein substrates targeted to Hsc66.     

<Superscript>1</Superscript>H, <Superscript>15</Superscript>N and <Superscript>13</Superscript>C resonance assignments of the J-domain of co-chaperone Sis1 from <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>

Protein folding in the cell is usually aided by molecular chaperones, from which the Hsp70 (Hsp?=?heat shock protein) family has many important roles, such as aiding nascent folding and participating in translocation. Hsp70 has ATPase activity which is stimulated by binding to the J-domain present in co-chaperones from the Hsp40 family. Hsp40s have many functions, as for instance the binding to partially folded proteins to be delivered to Hsp70. However, the presence of the J-domain characterizes Hsp40s or, by this reason, as J-proteins. The J-domain alone can stimulate Hsp70 ATPase activity. Apparently, it also maintains the same conformation as in the whole protein although structural information on full J-proteins is still missing. This work reports the ¹H, ¹⁵N and ¹³C resonance assignments of the J-domain of a Hsp40 from Saccharomyces cerevisiae, named Sis1. Secondary structure and order parameter prediction from chemical shifts are also reported. Altogether, the data show that Sis1 J-domain is highly structured and predominantly formed by α-helices, results that are in very good agreement with those previously reported for the crystallographic structure.     

Cytosolic and ER J-domains of mammalian and parasitic origin can functionally interact with DnaK

Both prokaryotic and eukaryotic cells contain multiple heat shock protein 40 (Hsp40) and heat shock protein 70 (Hsp70) proteins, which cooperate as molecular chaperones to ensure fidelity at all stages of protein biogenesis. The Hsp40 signature domain, the J-domain, is required for binding of an Hsp40 to a partner Hsp70, and may also play a role in the specificity of the association. Through the creation of chimeric Hsp40 proteins by the replacement of the J-domain of a prokaryotic Hsp40 (DnaJ), we have tested the functional equivalence of J-domains from a number of divergent Hsp40s of mammalian and parasitic origin (malarial Pfj1 and Pfj4, trypanosomal Tcj3, human ERj3, ERj5, and Hsj1, and murine ERj1). An in vivo functional assay was used to test the functionality of the chimeric proteins on the basis of their ability to reverse the thermosensitivity of a dnaJ cbpA mutant Escherichia coli strain (OD259). The Hsp40 chimeras containing J-domains originating from soluble (cytosolic or endoplasmic reticulum (ER)-lumenal) Hsp40s were able to reverse the thermosensitivity of E. coli OD259. In all cases, modified derivatives of these chimeric proteins containing an His to Gln substitution in the HPD motif of the J-domain were unable to reverse the thermosensitivity of E. coli OD259. This suggested that these J-domains exerted their in vivo functionality through a specific interaction with E. coli Hsp70, DnaK. Interestingly, a Hsp40 chimera containing the J-domain of ERj1, an integral membrane-bound ER Hsp40, was unable to reverse the thermosensitivity of E. coli OD259, suggesting that this J-domain was unable to functionally interact with DnaK. Substitutions of conserved amino acid residues and motifs were made in all four helices (I-IV) and the loop regions of the J-domains, and the modified chimeric Hsp40s were tested for functionality using the in vivo assay. Substitution of a highly conserved basic residue in helix II of the J-domain was found to disrupt in vivo functionality for all the J-domains tested. We propose that helix II and the HPD motif of the J-domain represent the fundamental elements of a binding surface required for the interaction of Hsp40s with Hsp70s, and that this surface has been conserved in mammalian, parasitic and bacterial systems.     

Mycoplasma pneumoniae J-domain protein required for terminal organelle function

The cell wall-less prokaryote Mycoplasma pneumoniae causes tracheobronchitis and primary atypical pneumonia in humans. Colonization of the respiratory epithelium requires proper assembly of a complex, multifunctional, polar terminal organelle. Loss of a predicted J-domain protein also having domains unique to mycoplasma terminal organelle proteins (TopJ) resulted in a non-motile, adherence-deficient phenotype. J-domain proteins typically stimulate ATPase activity of Hsp70 chaperones to bind nascent peptides for proper folding, translocation or macromolecular assembly, or to resolve stress-induced protein aggregates. By Western immunoblotting all defined terminal organelle proteins examined except protein P24 remained at wild-type levels in the topJ mutant; previous studies established that P24 is required for normal initiation of terminal organelle formation. Nevertheless, terminal organelle proteins P1, P30, HMW1 and P41 failed to localize to a cell pole, and when evaluated quantitatively, P30 and HMW1 foci were undetectable in >40% of cells. Complementation of the topJ mutant with the recombinant wild-type topJ allele largely restored terminal organelle development, gliding motility and cytadherence. We propose that this J-domain protein, which localizes to the base of the terminal organelle in wild-type M. pneumoniae , functions in the late stages of assembly, positioning, or both, of nascent terminal organelles.