Our results show that ERJ5
encodes a protein with an N-terminal signal sequence that is cleaved after translocation across the endoplasmic reticulum. Erj5p contains a lumenally exposed J domain, a single membrane spanning sequence, and a cytoplasmic carboxy-terminal tail. Protein localization analysis of Erj5p, showed that it colocalizes with Kar2p in the ER, confirming the assigned subcellular localization in a global analysis of yeast strains expressing GFP fusion proteins [36
]. These data support the identification and classification of Erj5p as the fourth DnaJ homologue with a J domain in the S. cerevisiae
Sequence comparison using available public databases identify Erj5p orthologues in all the fungal genomes sequenced to date, suggesting that Erj5 plays a role important enough to be preserved through evolution in yeast. All the sequences maintain the overall structure of Erj5p summarized in . The aligned sequences (http://db.yeastgenome.org/fungi/YFR041C.html
) show the highest level of conservation throughout the lumenal portion of the proteins but exhibit some variability at the carboxy-terminus in terms of amino acid identities and length.
The DnaJ homologues, possessing the highly conserved J domain, a signature of this family of proteins, have been classified into three groups based upon the domains shared with E. coli
DnaJ, the paradigmatic member of the group. Type I DnaJ proteins possess all three domains: the highly conserved J domain, the glycine-phenylalanine rich region, and a zinc finger-like domain. Type II J proteins lack the zinc finger-like domain, and Type III only possess the J domain [44
]. In the yeast ER, Scj1p is the only member of the Type I group. Jem1p, Sec63p and Erj5p are type III DnaJ homologues. Erj5p as well as Sec63p are integral membrane proteins whereas Scj1p and Jem1p are soluble proteins. These four DnaJ homologues have a J domain located in the lumen of the ER. The HPD motif, a hallmark in all J domains predicted to mediate interaction with Hsp70s, is present in Erj5p as well as in the other three J domains of the yeast ER ().
J-domain proteins are co-factors that regulate the ATP hydrolysis of their Hsp70 partners. Several lines of biochemical, functional and genetic evidences support the notion that Sec63p, Scj1p, and Jem1p act as co-factors of Kar2p, the main Hsp70 of the yeast ER [42
]. Having determined that Erj5p has a J-domain in the ER we tested for a role in Kar2p associated activities.
The essential Sec63p is an integral component of the yeast translocation complex [45
] that interacts with Kar2p to promote nascent chain transport into the ER. Erj5p would not appear to play a role similar to Sec63p, since we did not detect protein translocation defects by loss of Erj5p in wild-type strains, and the partial translocation block of a Δlhs1
mutant was not aggravated in a ΔlhsΔerj5
double mutant (data not shown). Therefore, Erj5p could function after the nascent chain has moved beyond the translocation channel.
A role for Erj5p in post-translocational Kar2p functions is supported by our results. Loss of the nonessential Scj1p and Jem1p DnaJ proteins that are required for Kar2p functions in the ER lumen, also yield translocation-proficient strains. Our genetic interaction data suggest that Kar2p may interact with Erj5p, in addition to interacting with Scj1p and Jem1p, in the ER lumen. Whereas Δscj1
and the kar2-159
mutation are synthetically lethal [24
], we found that although kar2-159Δerj5
strains are still viable, there is a synthetic negative interaction between Δerj5
and the kar2-159
mutation. Furthermore, the aggravation of the growth phenotype and sensitivity to ER stress that is generated by the loss of Erj5p in a Δscj1Δjem1
yeast strain would suggest a partial overlap of functions for Scj1p, Jem1p and Erj5p.
We next tested Erj5p in folding assays that disclosed a role for Scj1p and Jem1p in protein folding. Simultaneous loss of Scj1p and Jem1p causes a dramatic reduction of the transport rate of an unglycosylated mutant of carboxypeptidase Y (CPY) [11
], a soluble protein widely used to monitor protein folding in the yeast ER [9
]. We found that unglycosylated CPY is transported at similar rates in Δscj1Δjem1
mutant strains (data not shown). As for the group of genes with a role in protein maturation in the yeast ER, ERJ5
mRNA expression has been found elevated in a strain expressing a single misfolded secretory protein [6
]. However, a Δerj5
strain showed no changes in the secreted levels of a heterologous single chain antibody (scFv) compared to the wild-type strain (Xu, P., personal communication). It remains possible that the substrates tested may not be the optimal to reveal an ER folding delay caused by the loss of Erj5p. It is conceivable that whereas soluble lumenal Scj1p and Jem1p may participate with Kar2p in processes that take place in the ER lumen [11
], the topological restriction of the J domain of Erj5p to the ER membrane proximity might limit its function as a Kar2p cofactor to a more restricted environment or to a different and specific subset of untested folding proteins.
The co-existence in the ER of several members of the chaperone families with partially overlapping functions together with the ability of the UPR to compensate for the loss of components of the folding machinery by inducing a wide number of ER functions, make defining the specific role of each individual protein difficult. The wild-type growth rate of the Δscj1
mutants can be explained by the induction of the UPR [11
] that enhances expression of Kar2p and the others DnaJs of the ER. This explanation can be extended to understand the non-essential nature of Erj5p, an ER protein that, like Scj1p and Jem1p, is up-regulated by the UPR.
Without the UPR, key components of the ER folding machinery become limiting and the contribution of a gene to the ER homeostasis is evidenced. In a strain unable to induce the UPR (Δire1), loss of Erj5p causes a growth defect under normal conditions and is poorly tolerated under conditions that promote accumulation of misfolded proteins in the ER. This observation suggests that the Ire1p-dependent UPR is required to induce one or more factors that compensate for loss of Erj5p.
Our results show that ERJ5
mRNA is induced under conditions that promote stress in the ER, supporting a previous identification of the ERJ5
gene as an UPR target in a genomic analysis [21
]. Regulation of gene transcription by the UPR does not necessarily imply that loss of the gene will up-regulate the UPR in the cell. We have measured the UPR in the Δerj5
mutant by two independent approaches (KAR2
mRNA levels and activation of a GFP reporter sensor), and found that loss of Erj5p function leads to a small but significant chronic UPR induction. Constitutive induction of the UPR has been observed in yeast strains with mutations in genes whose functions are required for protein maturation in the ER: N-glycosylation [46
], chaperones [11
], and in genes required for ERAD that do not cause a detectable growth phenotype [21
]. The most likely explanation for a constitutive induction of the UPR is that a perturbation of the folding capacity of the ER is caused by loss of functions required for efficient performance of the ER biosynthetic machinery. Since the UPR is triggered by accumulation of misfolded proteins, constitutive induction of the UPR in these mutants is indicative of an altered folding capacity in the ER in absence of ER stress.
We extended our genetic analysis to get insight into the function of this new non-essential DnaJ of the yeast ER, combining the erj5
deletion with mutations in several ER genes whose functions are required for protein maturation, in which we had previously observed that loss of Scj1p caused growth defects at different temperatures whereas loss of Jem1p had very mild or no effect ([11
]and data not shown). We did not detect any growth phenotype associated to the loss of Erj5p in a N-glycosylation mutant lacking a non-essential subunit of the oligosaccharyltransferase (Δost3
), in a glucosidase1-deficient (Δgls1
) strain, or in a strain lacking calnexin (Δcne1
) (data not shown). These results are consistent with evidence that point to Scj1p as the main DnaJ homologue involved in ER protein folding. Jem1p, and also Erj5p, would be Kar2p regulatory co-factors whose absence results in a less pronounced perturbation of the ER folding capacity as indicated by a lower constitutive induction of KAR2
mRNA in the null strains, compared to Δscj1.
A critical function of the UPR is to reduce the lumenal concentration of misfolded proteins by either directly refolding proteins or removing them from the ER. Consistent with the dynamic requirements of the living cell, chaperones have been often found functionally involved in both processes. We have not tested for a possible role of Erj5p in ERAD. However, a number of groups have performed ERAD assays on strains in which the ERJ5 gene was deleted using a variety of ERAD substrates. The soluble substrate CPY* [47
], an integral membrane with the CPY* ERAD motif [48
], and the soluble mutant A1PiZ [49
]. No significant changes on the degradation rate of these proteins were detected by loss of Erj5p.
Although a global effect of Erj5p on ER protein maturation acting as a cofactor for Kar2p would be consistent with the genetic interactions detected, with the increased sensitivity to agents that promote accumulation of misfolded proteins in the ER, and with the effect on the UPR observed by loss of this J domain protein, it remains possible that Erj5p could perform a particular substrate-specific chaperone activity. In yeast, several genes involved in the biogenesis of specific secretory proteins have been identified, although the level at which they act is not fully understood in many cases [50
]. They may be required for folding and/or secretion, or they may act as specific quality control factors, since their deletion often result in the accumulation of specific substrate molecules in the ER. Although further studies will be required to define the exact function of Erj5p in the ER, the results of this work clearly establish that this DnaJ homologue is required for optimal performance of the yeast ER folding machinery.