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Peripheral myelin protein 22 (PMP22) and protein 0 (P0) are major peripheral myelin glycoproteins, and mutations in these two proteins are associated with hereditary demyelinating peripheral neuropathies. Calnexin, calreticulin, and ERp57 are critical components of protein quality control responsible for proper folding of newly synthesized glycoproteins. Here, using confocal microscopy, we show that cell surface targeting of P0 and PMP22 is not affected in the absence of the endoplasmic reticulum chaperones. However, the folding and function (adhesiveness) of PMP22 and P0, measured using the adhesion assay, are affected significantly in the absence of calnexin but not in the absence of calreticulin. Deficiency in oxidoreductase ERp57 results in impaired folding and function of P0, a disulfide bond-containing protein, but does not have any effect on folding or function of PMP22 (a protein that does not contain a disulfide bond). We concluded that calnexin and ERp57, but not calreticulin, play an important role in the biology of peripheral myelin proteins PMP22 and P0, and, consequently, these chaperones may contribute to the pathogenesis of peripheral neuropathies and the diversity of these neurological disorders.

Proper folding and maintenance of the native structure are central to protein function and are assisted by a family of proteins called chaperones. Calreticulin and calnexin are ER resident chaperones well conserved from worm to human. Calreticulin/calnexin knock-out mice exhibit a severe phenotype, whereas in Caenorhabditis elegans, calreticulin [crt-1(jh101)]- and calnexin [cnx-1(nr2009)]-null mutant worms exhibit only a mild phenotype, suggesting the possible existence of alternative chaperone machinery that can compensate for the deficiency of calreticulin and/or calnexin. In order to rapidly identify the compensatory chaperone components involved in this process, we analyzed the proteome of crt-1(jh101) mutants and [crt-1(jh101);cnx-1(nr2009)] double mutants. When grown at 20 degrees C, we found that five proteins were up-regulated and two proteins were down-regulated in crt-1(jh101) mutants; nine proteins were up-regulated and five proteins were down-regulated in [crt-1(jh101);cnx-1(nr2009)] double mutants. In addition, elevation of the cultivation temperature to 25 degrees C, which is still permissive to growth but causes specific defects in mutants, led to the identification of several additional proteins. Interestingly, the consistent increment of heat shock protein-70 family members (hsp70) together with protein disulfide isomerase (PDI) at all the examined conditions suggests the possible compensatory function imparted by hsp70 and PDI family members in the absence of calreticulin and/or calnexin.

Ca2+ responses to two chemoattractants, folate and cyclic AMP (cAMP), were assayed in Dictyostelium D. discoideum mutants deficient in one or both of two abundant Ca2+-binding proteins of the endoplasmic reticulum (ER), calreticulin and calnexin. Mutants deficient in either or both proteins exhibited enhanced cytosolic Ca2+ responses to both attractants. Not only were the mutant responses greater in amplitude, but they also exhibited earlier onsets, faster rise rates, earlier peaks, and faster fall rates. Correlations among these kinetic parameters and the response amplitudes suggested that key events in the Ca2+ response are autoregulated by the magnitude of the response itself, i.e., by cytosolic Ca2+ levels. This autoregulation was sufficient to explain the altered kinetics of the mutant responses: larger responses are faster in both mutant and wild-type cells in response to both folate (vegetative cells) and cAMP (differentiated cells). Searches of the predicted D. discoideum proteome revealed three putative Ca2+ pumps and four putative Ca2+ channels. All but one contained sequence motifs for Ca2+- or calmodulin-binding sites, consistent with Ca2+ signals being autoregulatory. Although cytosolic Ca2+ responses in the calnexin and calreticulin mutants are enhanced, the influx of Ca2+ from the extracellular medium into the mutant cells was smaller. Compared to wild-type cells, Ca2+ release from the ER in the mutants thus contributes more to the total cytosolic Ca2+ response while influx from the extracellular medium contributes less. These results provide the first molecular genetic evidence that release of Ca2+ from the ER contributes to cytosolic Ca2+ responses in D. discoideum.