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  • Title: Dissection of the translocation and chaperoning functions of yeast BiP/Kar2p in vivo.
    Author: Holkeri H, Paunola E, Jämsä E, Makarow M.
    Journal: J Cell Sci; 1998 Mar; 111 ( Pt 6)():749-57. PubMed ID: 9472003.
    Abstract:
    We used the rat nerve growth factor receptor ectodomain (NGFRe) and Escherichia coli ss-lactamase to dissect the functions of Saccharomyces cerevisiae BiP/Kar2p in vivo. Both were fused to the Hsp150Delta-polypeptide, which promotes proper folding of heterologous proteins which otherwise are misfolded in the yeast ER. Hsp150Delta-NGFRe and Hsp150Delta-beta-lactamase acquired disulfides and were properly folded and ONcreted to the culture medium. When disulfide formation was prevented by incubating cells with dithiothreitol (DTT), Hsp150Delta-NGFRe remained in the endoplasmic reticulum (ER). The occupancy of an otherwise partially used N-glycosylation site of reduced NGFRe was complete suggesting that, normally, folding and disulfide formation occurred as rapidly as N-glycosylation. Removal of DTT resulted in remarkably rapid disulfide formation and secretion, suggesting only mild conformational distortion of reduced NGFRe. In contrast, reduced Hsp150(Delta)-ss-lactamase was severely misfolded and attained a secretion competent conformation more slowly after reoxidation. When kar2-159 cells were incubated at permissive temperature 24 degrees C with DTT, the reporter proteins were retained in the ER. After shift of the cells to 34 degrees C to inactivate BiP/Kar2p irreversibly, and subsequent removal of DTT, most pre-accumulated Hsp150Delta-NGFRe was rapidly secreted, whereas Hsp150Delta-beta-lactamase was secretion incompetent. Thus, Hsp150Delta-NGFRe did not require BiP/Kar2p for conformational maturation, though translocation was dependent on BiP/Kar2p. Apparently proteins differ in their post-translocational requirements for BiP/Kar2p, indicating that translocation and chaperoning are distinct functions.
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