Intracellular translocation and stability of apolipoprotein B are inversely proportional to the length of the nascent polypeptide |
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Authors: | D Cavallo RS McLeod D Rudy A Aiton Z Yao K Adeli |
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Affiliation: | Department of Chemistry and Biochemistry, University of Windsor, Windsor, Ontario, Canada N9B 3P4. |
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Abstract: | We have studied the relationship between the length of apolipoprotein B (apoB) and its intracellular translocation and stability using McArdle RH7777 (McA-RH7777) cells expressing recombinant human apoB variants, ranging in size from B15 to B100. The translocational status of apoB was assessed based on trypsin sensitivity of apoB using isolated microsomes as well as permeabilized cells. In isolated microsomes, shorter apoB variants (=B48) were 75-100% resistant to exogenous trypsin digestion, whereas apoB variants larger than B48 were less than 40% trypsin-resistant. Experiments with hepatic microsomes isolated from rat or transgenic mice expressing human B48 and B100 also confirmed the high trypsin accessibility of B100 compared with B48. In permeabilized cells, apoB variants shorter than B48 were relatively resistant to exogenous trypsin (percentage of trypsin-resistant apoB greater than 70%) in contrast to recombinant human B72 and B100, which were only 55 and 42% trypsin-resistant, respectively. The trypsin sensitivity of human B100 was comparable with that of endogenous rat B100 in McA-RH7777 cells as well as endogenous B100 in HepG2 cells (percentages of trypsin-resistant cells were as follows: for human B100 construct, 42 +/- 7.5%; for endogenous McA-RH7777 B100, 52 +/- 2.9%; and for endogenous HepG2 B100, 46 +/- 6.3%). Overall, an inverse correlation between the length of apoB and its resistance to exogenous trypsin was evident irrespective of the model system examined. An inverse relationship was also observed between the size of apoB and its co-translational resistance to proteasomal degradation. Truncated apoB constructs were relatively insensitive to proteasome inhibition by MG132 co-translationally (during the pulse) compared with the full-length B100, which was highly sensitive (apoB recovered in the presence of MG132 as a percentage of control was as follows: B15, 127%; B29, 94%; B48, 110%; B72, 140%; B100, 282%). Post-translationally (over a 2-h chase), a similar inverse relationship was found, with B100 being the least stable in comparison with truncated apoB variants. In summary, as the size of the nascent apoB chain increases, there appears to be a greater cytosolic exposure of the polypeptide, leading to a higher sensitivity to proteasomal degradation. |
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