Disease relevance of KAR2

• This provides the first evidence that KAR2 facilitates the assembly of an oligomeric protein in yeast and thus implicates KAR2 as a 'molecular chaperone'. The possible mechanisms of enterotoxoid assembly in E. coli and S. cerevisiae are discussed [1].
• We report here that yeast expressing the cystic fibrosis transmembrane conductance regulator (CFTR) concentrate the protein at defined sites in the ER membrane that are not necessarily enriched for the ER molecular chaperone BiP [2].

High impact information on KAR2

• Multiple BiP molecules associate with each translocation substrate following interaction with the J domain of the Sec63p component of the Sec complex [3].
• Extraction of soluble lumenal proteins from microsomes and reconstitution with purified proteins demonstrate, by fluorescence collisional quenching, that BiP seals the lumenal end of this pore [4].
• Several yeast mutants defective in this pathway map to the ERN1 gene, which protects cells from lethal consequences of stress by signaling for increased expression of BiP and other ER proteins [5].
• These results suggest that Sec61p is directly involved in translocation and that BiP acts at two stages of the translocation cycle [6].
• The gene encoding yeast BiP is essential for cell growth and, unexpectedly, is identical to the recently cloned KAR2 gene [7].

Biological context of KAR2

• As a first step toward identifying cell components important in folding of the nascent ATPase, we have used the dual assays to examine the role of KAR2, encoding the yeast homolog of immunoglobulin heavy chain binding protein/78-kDa glucose-regulated protein, and SEC65, encoding a subunit of the yeast signal recognition particle [8].
• However, introduction of the wild-type KAR2 gene on a plasmid into the kar2-1 mutant completely suppressed the inhibition of Lipo-EtxB assembly [1].
• All human cDNAs and yeast multicopy suppressors, which had been isolated as suppressors for the ire15 mutation, were able to suppress the inositol-auxotrophic phenotype but not the defect in KAR2 induction of the hac1-disrupted strain [9].
• The tunicamycin sensitivity of (delta)ire1 cells was also suppressed by extra expression of KAR2, suggesting that BiP plays a principal role in protecting cell growth against misfolded proteins accumulated in the ER [10].
• 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 [11].

Anatomical context of KAR2

• Although mutation of KAR2 caused defective translocation of several secretory precursors into the endoplasmic reticulum lumen, ATPase folding and intracellular transport were unperturbed [8].
• Folding and intracellular transport of the yeast plasma-membrane H(+)-ATPase: effects of mutations in KAR2 and SEC65 [8].
• 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 [11].
• These results show that loss of functional Ssa1p from the cytosol up-regulates KAR2 gene expression through an HSE-mediated pathway and also support the idea that SSA1 gene expression is autoregulated [12].
• We now show that the cne1Delta and two kar2 mutant alleles exhibit a synthetic interaction and that the export and degradation of pro-alpha factor is defective in kar2 mutant microsomes [13].

Associations of KAR2 with chemical compounds

• The Saccharomyces cerevisiae IRE1 gene, encoding a putative receptor-type protein kinase, is known to be required for inositol prototrophy and for the induction of a chaperon molecule, BiP, encoded by KAR2, under stress conditions such as tunicamycin addition [14].
• The genetic interaction of kar2 and wbp1 mutations. Distinct functions of binding protein BiP and N-linked glycosylation in the processing pathway of secreted proteins in Saccharomyces cerevisiae [15].
• Under the latter conditions, subcellular fractionation on sucrose gradients revealed, in addition to the expected plant PMA2 peak linked to the plasma membrane fraction, low density peak containing PMA2 and KAR2, an endoplasmic reticulum marker [16].
• However, we found an increase in the expression of KAR2 (karyogamy 2) gene, a well-known marker of ER (endoplasmic reticulum) stress and also observed HAC1 cleavage in homocysteine- and cysteinetreated cells, which indicates that homocysteine- and cysteine-mediated growth defect may probably be attributed to ER stress [17].
• For the membrane fusion step, one required component, the ER luminal protein Kar2p (BiP), has been identified [18].

Physical interactions of KAR2

• ROT1 genetically interacted with several ER chaperone genes including KAR2, and the rot1-2 mutation triggered the unfolded protein response [19].
• In the presence of ATP, under conditions in which BiP can bind to Sec63p, the secretory precursor passes from the cytosol into the lumen through a membrane channel formed by Sec61p [20].
• We speculate that recognition of unfolded proteins is based on their competition with Ire1 for binding with BiP/Kar2 [21].
• Deletion of SSI1 shows a complex pattern of genetic interactions with various conditional alleles of KAR2, ranging from synthetic lethality to synthetic rescue [22].

Regulatory relationships of KAR2

• Genetic evidence for a role of BiP/Kar2 that regulates Ire1 in response to accumulation of unfolded proteins [21].
• Finally, we found that the soluble Sec63p lumenal domain inhibited efficient precursor import into proteoliposomes reconstituted so as to incorporate both BiP and the fusion protein [23].
• 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 [24].

Other interactions of KAR2

• A Sec63p-BiP complex from yeast is required for protein translocation in a reconstituted proteoliposome [11].
• Genetic analyses involving the UPR target gene KAR2 and the UPR regulator IRE1 revealed that autodiploidization associated with hac1 mutants is a consequence of its role in the UPR pathway [25].
• Overexpression of both S. cerevisiae HAC1 and T. reesei hac1 caused an increase in the expression of the known UPR target gene KAR2 at early time points during cultivation [26].
• We show that a combination of kar2 and wbp1 mutations results in a synthetic phenotype with a strongly reduced growth rate at the permissive temperature [15].
• As with KAR2, FKB2 mRNA levels are also elevated by heat shock [27].

Analytical, diagnostic and therapeutic context of KAR2

• Using the mRNA differential display technique and Western blot analysis, the present study demonstrates that induction of KAR2 occurs when misfolded membrane-bound cytochrome P450, mutated in its cytosolically exposed domain, is expressed in Saccharomyces cerevisiae [28].
• Double-label immunofluorescence experiments show that the HlyB structures also contain the ER binding protein (BiP), the product of the KAR2 gene [29].
• According to co-immunoprecipitation experiments, immuno-globulin-binding protein (BiP/Kar2p) was associated with the fusion protein [30].
• Freeze-thaw treatment, hydroxyapatite high pressure liquid chromatography, and ATP-agarose column chromatography of crude extract yielded homogeneous BiP/Kar2 protein (including less than 0.2% of degradative derivative) with a 430-fold purification and 28% recovery [31].
• Dissection of the translocation and chaperoning functions of yeast BiP/Kar2p in vivo [32].

References