Differential regulation of MAP2 by phosphorylation events in proline-rich versus C-terminal domains.

MAP2 is a critical cytoskeletal regulator in neurons. The phosphorylation of MAP2 (MAP2-P) is well known to regulate core functions of MAP2, including microtubule (MT)/actin binding and facilitation of tubulin polymerization. However, site-specific studies of MAP2-P function in regions outside of the MT-binding domain (MTBD) are lacking. We previously identified a set of MAP2 phosphopeptides which are differentially expressed and predominantly increased in the cortex of individuals with schizophrenia relative to nonpsychiatric comparison subjects. The phosphopeptides originated not from the MTBD, but from the flanking proline-rich and C-terminal domains of MAP2. We sought to understand the contribution of MAP2-P at these sites on MAP2 function. To this end, we isolated a series of phosphomimetic MAP2C constructs and subjected them to cell-free tubulin polymerization, MT-binding, actin-binding, and actin polymerization assays. A subset of MAP2-P events significantly impaired these functions, with the two domains displaying different patterns of MAP2 regulation: proline-rich domain mutants T293E and T300E impaired MT assembly and actin-binding affinity but did not affect MT-binding, while C-terminal domain mutants S426E and S439D impaired all three functions. S443D also impaired MT assembly with minimal effects on MT- or actin-binding. Using heterologous cells, we also found that S426E but not T293E had a lower capability for process formation than the wild-type protein. These findings demonstrate the functional utility of MAP2-P in the proline-rich and C-terminal domains and point to distinct, domain-dependent regulations of MAP2 function, which can go on to affect cellular morphology.

Molecular chaperones and substrate ubiquitination control the efficiency of endoplasmic reticulum-associated degradation.

The endoplasmic reticulum (ER) must contend with a large protein flux, which is especially notable in cells dedicated to secreting hormone-regulated gene products. Because of the complexity of the protein folding pathway and the potential for genetic or stochastic errors, a significant percentage of these nascent secreted proteins fail to acquire their native conformations. If these species cannot be cleared from the ER, they may aggregate, which leads to cell death. To lessen the effects of potentially toxic polypeptides, aberrant ER proteins are destroyed via a process known as ER-associated degradation (ERAD). ERAD substrates are selected by molecular chaperones and chaperone-like proteins, and prior to degradation most substrates are ubiquitin-modified. Together with the unfolded protein response, the ERAD pathway is a critical component of the protein quality control machinery in the ER. Although emerging data continue to link ERAD with human diseases, most of our knowledge of this pathway arose from studies using a model eukaryote, the yeast Saccharomyces cerevisiae. In this review, we will summarize the discoveries that led to our current understanding of this pathway, focusing primarily on experiments in yeast. We will also indicate links between ERAD and disease and emphasize future research avenues.

ER-associated and proteasomemediated protein degradation: how two topologically restricted events came together.

A protein-degradation pathway associated with the endoplasmic reticulum (ER) can selectively remove polypeptides from the secretory pathway. The mechanisms of this ER-associated protein degradation were obscure, but recent studies using both yeast and mammalian cells have indicated that substrates for degradation are targeted to the cytosol where proteolysis is catalysed by the proteasome. The degradation process is now known to comprise at least three distinct events: first, recognition of a polypeptide for degradation; second, efflux of this substrate from the ER to the cytosol; and, finally, degradation by the proteasome. This review summarizes recent advances in understanding how each of these steps is achieved.

A Sec63p-BiP complex from yeast is required for protein translocation in a reconstituted proteoliposome.

Reconstituted proteoliposomes derived from solubilized yeast microsomes are able to translocate a secreted yeast mating pheromone precursor (Brodsky, J. L., S. Hamamoto, D. Feldheim, and R. Schekman. 1993. J. Cell Biol. 120:95-107). Reconstituted proteoliposomes prepared from strains with mutations in the SEC63 or KAR2 genes are defective for translocation; the kar2 defect can be overcome by the addition of purified BiP (encoded by the KAR2 gene). We now show that addition of BiP to wild-type reconstituted vesicles increases their translocation efficiency three-fold. To identify other ER components that are required for translocation, we purified a microsomal membrane protein complex that contains Sec63p. We found that the complex also includes BiP, Sec66p (gp31.5), and Sec67p (p23). The Sec63p complex restores translocation activity to reconstituted vesicles that are prepared from a sec63-1 strain, or from cells in which the SEC66 or SEC67 genes are disrupted. BiP dissociates from the complex when the purification is performed in the presence of ATP gamma S or when the starting membranes are from yeast containing the sec63-1 mutation. We conclude that the purified Sec63p complex is active and required for protein translocation, and that the association of BiP with the complex may be regulated in vivo.

Assembly of ER-associated protein degradation in vitro: dependence on cytosol, calnexin, and ATP.

To investigate the mechanisms of ER-associated protein degradation (ERAD), this process was reconstituted in vitro. Established procedures for post-translational translocation of radiolabeled prepro-alpha factor into isolated yeast microsomes were modified to inhibit glycosylation and to include a posttranslocation "chase" incubation period to monitor degradation. Glycosylation was inhibited with a glyco-acceptor peptide to compete for core carbohydrates, or by using a radio-labeled alpha factor precursor that had been genetically engineered to eliminate all three glycosylation sites. Inhibition of glycosylation led to the production of unglycosylated pro-alpha factor (p alpha F), a processed form of the alpha factor precursor shown to be a substrate of ERAD in vivo. With this system, both glycosylated and unglycosylated forms of pro-alpha factor were stable throughout a 90-min chase incubation. However, the addition of cytosol to the chase incubation reaction induced a selective and rapid degradation of p alpha F. These results directly reflect the behavior of alpha factor precursor in vivo; i.e., p alpha F is a substrate for ERAD, while glycosylated pro-alpha factor is not. Heat inactivation and trypsin treatment of cytosol, as well as addition of ATP gamma S to the chase incubations, led to a stabilization of p alpha F. ERAD was observed in sec12 microsomes, indicating that export of p alpha F via transport vesicles was not required. Furthermore, p alpha F but not glycosylated pro-alpha factor was found in the supernatant of the chase incubation reactions, suggesting a specific transport system for this ERAD substrate. Finally, the degradation of p alpha F was inhibited when microsomes from a yeast strain containing a disrupted calnexin gene were examined. Together, these results indicate that cytosolic protein factor(s), ATP hydrolysis, and calnexin are required for ER-associated protein degradation in yeast, and suggest the cytosol as the site for degradation.

Reconstitution of protein translocation from solubilized yeast membranes reveals topologically distinct roles for BiP and cytosolic Hsc70.

We reconstituted prepro-alpha-factor translocation and signal peptide processing using a yeast microsomal detergent soluble fraction formed into vesicles with soybean phospholipids. Reconstituted translocation required ATP, and was deficient when sec63 and kar2 (BiP) mutant cells were used as a source of membranes. Normal translocation was observed with vesicles reconstituted from a mixture of pure wild-type yeast BiP and a soluble fraction of kar2 mutant membranes. Two other heat-shock cognate (hsc) 70 homologs, yeast cytosolic hsc70 (Ssalp) and E. coli dnaK protein did not replace BiP. Conversely, BiP was not active under conditions where translocation into native ER vesicles required cytosolic hsc70. We conclude that cytosolic hsc70 and BiP serve noninterchangeable roles in polypeptide translocation, possibly because distinct, asymmetrically oriented membrane proteins are required to recruit each protein to opposing surfaces of the ER membrane.

Molecular chaperones in the yeast endoplasmic reticulum maintain the solubility of proteins for retrotranslocation and degradation.

Endoplasmic reticulum (ER)-associated degradation (ERAD) is the process by which aberrant proteins in the ER lumen are exported back to the cytosol and degraded by the proteasome. Although ER molecular chaperones are required for ERAD, their specific role(s) in this process have been ill defined. To understand how one group of interacting lumenal chaperones facilitates ERAD, the fates of pro-alpha-factor and a mutant form of carboxypeptidase Y were examined both in vivo and in vitro. We found that these ERAD substrates are stabilized and aggregate in the ER at elevated temperatures when BiP, the lumenal Hsp70 molecular chaperone, is mutated, or when the genes encoding the J domain-containing proteins Jem1p and Scj1p are deleted. In contrast, deletion of JEM1 and SCJ1 had little effect on the ERAD of a membrane protein. These results suggest that one role of the BiP, Jem1p, and Scj1p chaperones is to maintain lumenal ERAD substrates in a retrotranslocation-competent state.

Post-translational protein translocation: not all hsc70s are created equal.

Hsc70s in the yeast endoplasmic reticulum (ER) and mitochondria interact with membrane-associated components of the translocation machinery and are required for post-translational protein import. Although it has been proposed that the mitochondrial and ER machines function similarly, a variety of experiments suggest that BiP, the ER hsc70, might play a more elaborate role.

Heat shock protein 101 effects in A. thaliana: genetic variation, fitness and pleiotropy in controlled temperature conditions.

The Hsp100/ClpB heat shock protein family is ancient and required for high temperature survival, but natural variation in expression and its phenotypic effects is unexplored in plants. In controlled environment experiments, we examined the effects of variation in the Arabidopsis cytosolic AtHsp101 (hereafter Hsp101). Ten wild-collected ecotypes differed in Hsp101 expression responses across a 22 to 40 degrees C gradient. Genotypes from low latitudes expressed the least Hsp101. We tested fitness and pleiotropic consequences of varying Hsp101 expression in 'control' vs. mild thermal stress treatments (15/25 degrees C D/N vs. 15/25 degrees D/N plus 3 h at 35 degrees C 3 days/week). Comparing wild type and null mutants, wt Columbia (Col) produced approximately 33% more fruits compared to its Hsp101 homozygous null mutant. There was no difference between Landsberg erecta null mutant NIL (Ler) and wt Ler; wt Ler showed very low Hsp101 expression. In an assay of six genotypes, fecundity was a saturating function of Hsp101 content, in both experimental treatments. Thus, in addition to its essential role in acquired thermal tolerance, Hsp101 provides a substantial fitness benefit under normal growth conditions. Knocking out Hsp101 decreased fruit production, days to germination and days to bolting, total dry mass, and number of inflorescences; it increased transpiration rate and allocation to root mass. Root : total mass ratio decayed exponentially with Hsp101 content. This study shows that Hsp101 expression is evolvable in natural populations. Our results further suggest that Hsp101 is primarily an emergency high-temperature tolerance mechanism, since expression levels are lower in low-latitude populations from warmer climates. Hsp101 expression appears to carry an important trade-off in reduced root growth. This trade-off may select for suppressed expression under chronically high temperatures.

Species-specific elements in the large T-antigen J domain are required for cellular transformation and DNA replication by simian virus 40.

The J domain of simian virus 40 (SV40) large T antigen is required for efficient DNA replication and transformation. Despite previous reports demonstrating the promiscuity of J domains in heterologous systems, results presented here show the requirement for specific J-domain sequences in SV40 large-T-antigen-mediated activities. In particular, chimeric-T-antigen constructs in which the SV40 T-antigen J domain was replaced with that from the yeast Ydj1p or Escherichia coli DnaJ proteins failed to replicate in BSC40 cells and did not transform REF52 cells. However, T antigen containing the JC virus J domain was functional in these assays, although it was less efficient than the wild type. The inability of some large-T-antigen chimeras to promote DNA replication and elicit cellular transformation was not due to a failure to interact with hsc70, since a nonfunctional chimera, containing the DnaJ J domain, bound hsc70. However, this nonfunctional chimeric T antigen was reduced in its ability to stimulate hsc70 ATPase activity and unable to liberate E2F from p130, indicating that transcriptional activation of factors required for cell growth and DNA replication may be compromised. Our data suggest that the T-antigen J domain harbors species-specific elements required for viral activities in vivo.

The amino-terminal transforming region of simian virus 40 large T and small t antigens functions as a J domain.

Simian virus 40 (SV40) encodes two proteins, large T antigen and small t antigen that contribute to virus-induced tumorigenesis. Both proteins act by targeting key cellular regulatory proteins and altering their function. Known targets of the 708-amino-acid large T antigen include the three members of the retinoblastoma protein family (pRb, p107, and p130), members of the CBP family of transcriptional adapter proteins (cap-binding protein [CBP], p300, and p400), and the tumor suppressor p53. Small t antigen alters the activity of phosphatase pp2A and transactivates the cyclin A promoter. The first 82 amino acids of large T antigen and small t antigen are identical, and genetic experiments suggest that an additional target(s) important for transformation interacts with these sequences. This region contains a motif similar to the J domain, a conserved sequence found in the DnaJ family of molecular chaperones. We show here that mutations within the J domain abrogate the ability of large T antigen to transform mammalian cells. To examine whether a purified 136-amino-acid fragment from the T antigen amino terminus acts as a DnaJ-like chaperone, we investigated whether this fragment stimulates the ATPase activity of two hsc70s and discovered that ATP hydrolysis is stimulated four- to ninefold. In addition, ATPase-defective mutants of full-length T antigen, as well as wild-type small t antigen, stimulated the ATPase activity of hsc70. T antigen derivatives were also able to release an unfolded polypeptide substrate from an hsc70, an activity common to DnaJ chaperones. Because the J domain of T antigen plays essential roles in viral DNA replication, transcriptional control, virion assembly, and tumorigenesis, we conclude that this region may chaperone the rearrangement of multiprotein complexes.

Specific molecular chaperone interactions and an ATP-dependent conformational change are required during posttranslational protein translocation into the yeast ER.

The posttranslational translocation of proteins across the endoplasmic reticulum (ER) membrane in yeast requires ATP hydrolysis and the action of hsc70s (DnaK homologues) and DnaJ homologues in both the cytosol and ER lumen. Although the cytosolic hsc70 (Ssa1p) and the ER lumenal hsc70 (BiP) are homologous, they cannot substitute for one another, possibly because they interact with specific DnaJ homologues on each side of the ER membrane. To investigate this possibility, we purified Ssa1p, BiP, Ydj1p (a cytosolic DnaJ homologue), and a GST-63Jp fusion protein containing the lumenal DnaJ region of Sec63p. We observed that BiP, but not Ssa1p, is able to associate with GST-63Jp and that Ydj1p stimulates the ATPase activity of Ssa1p up to 10-fold but increases the ATPase activity of BiP by <2-fold. In addition, Ydj1p and ATP trigger the release of an unfolded polypeptide from Ssa1p but not from BiP. To understand further how BiP drives protein translocation, we purified four dominant lethal mutants of BiP. We discovered that each mutant is defective for ATP hydrolysis, fails to undergo an ATP-dependent conformational change, and cannot interact with GST-63Jp. Measurements of protein translocation into reconstituted proteoliposomes indicate that the mutants inhibit translocation even in the presence of wild-type BiP. We conclude that a conformation- and ATP-dependent interaction of BiP with the J domain of Sec63p is essential for protein translocation and that the specificity of hsc70 action is dictated by their DnaJ partners.

Hsp70 molecular chaperone facilitates endoplasmic reticulum-associated protein degradation of cystic fibrosis transmembrane conductance regulator in yeast.

Membrane and secretory proteins fold in the endoplasmic reticulum (ER), and misfolded proteins may be retained and targeted for ER-associated protein degradation (ERAD). To elucidate the mechanism by which an integral membrane protein in the ER is degraded, we studied the fate of the cystic fibrosis transmembrane conductance regulator (CFTR) in the yeast Saccharomyces cerevisiae. Our data indicate that CFTR resides in the ER and is stabilized in strains defective for proteasome activity or deleted for the ubiquitin-conjugating enzymes Ubc6p and Ubc7p, thus demonstrating that CFTR is a bona fide ERAD substrate in yeast. We also found that heat shock protein 70 (Hsp70), although not required for the degradation of soluble lumenal ERAD substrates, is required to facilitate CFTR turnover. Conversely, calnexin and binding protein (BiP), which are required for the proteolysis of ER lumenal proteins in both yeast and mammals, are dispensable for the degradation of CFTR, suggesting unique mechanisms for the disposal of at least some soluble and integral membrane ERAD substrates in yeast.

Recognition and delivery of ERAD substrates to the proteasome and alternative paths for cell survival.

Endoplasmic reticulum-associated protein degradation (ERAD) is a protein quality control mechanism that minimizes the detrimental effects of protein misfolding in the secretory pathway. Molecular chaperones and ER lumenal lectins are essential components of this process because they maintain the solubility of unfolded proteins and can target ERAD substrates to the cytoplasmic proteasome. Other factors are likely required to aid in the selection of ERAD substrates, and distinct proteinaceous machineries are required for substrate retrotranslocation/dislocation from the ER and proteasome targeting. When the capacity of the ERAD machinery is exceeded or compromised, multiple degradative routes can be enlisted to prevent the detrimental consequences of ERAD substrate accumulation, which include cell death and disease.

Translocation of proteins across the endoplasmic reticulum membrane.

Secretory protein biogenesis begins with the insertion of a preprotein into the lumen of the endoplasmic reticulum (ER). This insertion event, known as ER protein translocation, can occur either posttranslationally, in which the preprotein is completely synthesized on cytosolic ribosomes before being translocated, or cotranslationally, in which membrane-associated ribosomes direct the nascent polypeptide chain into the ER concomitant with polypeptide elongation. In either case, preproteins are targeted to the ER membrane through specific interactions with cytosolic and/or ER membrane factors. The preprotein is then transferred to a multiprotein translocation machine in the ER membrane that includes a pore through which the preprotein passes into the ER lumen. The energy required to drive protein translocation may derive either from the coupling of translation to translocation (during cotranslational translocation) or from ER lumenal molecular chaperones that may harness the preprotein or regulate the translocation machinery (during posttranslational translocation).

Mutation of the ATP-binding pocket of SSA1 indicates that a functional interaction between Ssa1p and Ydj1p is required for post-translational translocation into the yeast endoplasmic reticulum.

The translocation of proteins across the yeast ER membrane requires ATP hydrolysis and the action of DnaK (hsp70) and DnaJ homologues. In Saccharomyces cerevisiae the cytosolic hsp70s that promote post-translational translocation are the products of the Ssa gene family. Ssa1p maintains secretory precursors in a translocation-competent state and interacts with Ydj1p, a DnaJ homologue. Although it has been proposed that Ydj1p stimulates the ATPase activity of Ssa1p to release preproteins and engineer translocation, support for this model is incomplete. To this end, mutations in the ATP-binding pocket of SSA1 were constructed and examined both in vivo and in vitro. Expression of the mutant Ssa1p's slows wild-type cell growth, is insufficient to support life in the absence of functional Ssa1p, and results in a dominant effect on post-translational translocation. The ATPase activity of the purified mutant proteins was not enhanced by Ydj1p and the mutant proteins could not bind an unfolded polypeptide substrate. Our data suggest that a productive interaction between Ssa1p and Ydj1p is required to promote protein translocation.

Isolation and characterization of a DnaJ-like protein in rats: the C-terminal 10-kDa domain of hsc70 is not essential for stimulating the ATP-hydrolytic activity of hsc70 by a DnaJ-like protein.

A DnaJ-like protein, RDJ1, was isolated from a rat brain cDNA library. The protein is predicted to have 397 amino acid residues and shares 99% identity to that of HDJ2, a human DnaJ-like protein. RDJ1 was also shown to rescue the temperature-sensitive lethality of a strain containing a mutated cytosolic DnaJ in yeast, ydj1-151. Fragments containing the J-domain of RDJ1 either with or without the G/F motif were expressed in Escherichia coli. The purified proteins stimulated the ATPase activity of hsc70 and of the 60-kDa N-terminal fragment of hsc70. These results imply that RDJ1 can interact with the N-terminal 60-kDa fragment of hsc70 to activate ATP hydrolysis by hsc70.

Mitochondrial Hsp70 cannot replace BiP in driving protein translocation into the yeast endoplasmic reticulum.

To determine whether mitochondrial hsp70 (mHsp70) could substitute for the endoplasmic retuculum (ER) Hsp70 (BiP) during protein translocation, we assembled ER-derived reconstituted proteoliposomes supplemented with either protein. We found that only BiP restored translocation in kar2 mutant vesicles and stimulated translocation approximately 3-fold in wild type proteoliposomes. mHsp70 associated poorly with both a BiP binding (DnaJ) domain of Sec63p and an ER precursor, and its ATPase activity was poorly enhanced upon incubation with the DnaJ domain. In contrast, BiP bound to the Sec63p-DnaJ domain in an ATP-dependent manner and its ATPase activity was stimulated significantly by this polypeptide. We conclude that mHsp70 is unable to support protein translocation into the ER because it fails to associate productively with Sec63p and a precursor.

Selectivity of the molecular chaperone-specific immunosuppressive agent 15-deoxyspergualin: modulation of Hsc70 ATPase activity without compromising DnaJ chaperone interactions.

The immunosuppressive and cytostatic agent 15-deoxyspergualin (DSG) binds to the Hsc70 class of molecular chaperones with a K(D) = 4 microM. Because Hsc70s represent a diverse group of cellular effectors and because Hsc70 function frequently requires a DnaJ molecular chaperone, the specificity of DSG for different Hsc70s and the ability of DSG to block the productive interaction between an Hsc70 and its DnaJ partner were examined. DSG stimulated the ATPase activity of a mammalian and yeast cytosolic Hsc70 from 20 to 40%, but was unable to elicit such a response in a homologous Hsc70, Binding Protein (BiP), that resides in the lumen of the endoplasmic reticulum. In addition, the DnaJ-stimulated Hsc70 ATPase activity and the DnaJ-mediated release of an unfolded polypeptide from an Hsc70 were unaffected by DSG. These results indicate that Hsc70s exhibit substrate selectivity for DSG and that DSG does not compromise Hsc70 functions that require DnaJs. Thus, the immunosuppressive and cytostatic effects of DSG may be specific for a subset of cellular Hsc70s and confined to DnaJ-independent Hsc70-mediated activities.

The yeast Saccharomyces cerevisiae does not sequester chloride but can express a functional mammalian chloride channel.

Chloride uptake into yeast was measured as a function of pH. A small amount of uptake was seen at pH values of 3.0 and 4.0; at pH 6.0 chloride uptake was substantially less than the uptake of phosphate and rubidium. Because chloride uptake is inefficient, we expressed the putative mammalian chloride channel, pI(Cln), in yeast and observed a chloride-selective current when total membrane protein was reconstituted into lipid bilayers. The current was inhibited by a specific chloride channel blocker, 5-nitro-2-(3-phenylpropylamino)-benzoic acid. These results suggest that yeast may serve as a means to characterize chloride channels from other organisms.