Analysis of apolipoprotein E nuclear localization using green fluorescent protein and biotinylation approaches.
Previous results indicate that apoE (apolipoprotein E) may be associated with the nucleus in specific cell types, particularly under stress conditions such as serum starvation. In addition, nuclear apoE localization in ovarian cancer was recently shown to be correlated with patient survival. In order to better understand the factors associated with apoE nuclear localization, we examined intracellular apoE trafficking using live-cell imaging of CHO (Chinese-hamster ovary) cells that constitutively expressed apoE-GFP (green fluorescent protein). In addition, we used biotinylated apoE (in a lipid-free state and as a lipidated discoidal complex) to track the uptake and potential nuclear targeting of exogenous apoE. Our results indicate that a small proportion of apoE-GFP is detected in the nucleus of living apoE-GFP-expressing CHO cells and that the level of apoE-GFP in the nucleus is increased with serum starvation. Exposure of control CHO cells to exogenous apoE-GFP did not result in nuclear apoE-GFP localization in the recipient cells. Similarly, biotinylated apoE did not reach the nucleus of control CHO cells or SK-N-SH neurons. In contrast, when biotinylated apoE was delivered to recipient cells as a lipidated apoE disc, apoE was detected in the nucleus, suggesting that the lipoprotein complex alters the intracellular degradation or trafficking of apoE. Biotinylated apoE discs containing each of the three common human apoE isoforms (E2, E3 and E4) were also tested for nuclear trafficking. All three apoE isoforms were equally detected in the nucleus. These studies provide new evidence that apoE may be targeted to the nucleus and shed light on factors that regulate this process.
Kim
Woojin S
WS
Prince of Wales Medical Research Institute, Randwick, NSW 2031, Australia.
Elliott
David A
DA
Kockx
Maaike
M
Kritharides
Leonard
L
Rye
Kerry-Anne
KA
Jans
David A
DA
Garner
Brett
B
D021581:Active Transport, Cell Nucleus; D000818:Animals; D001057:Apolipoproteins E / Q000032:analysis* / Q000235:genetics / Q000378:metabolism*; D019921:Biotinylation; D002460:Cell Line; D002467:Cell Nucleus / Q000378:metabolism; D006224:Cricetinae; D005786:Gene Expression Regulation; D017930:Genes, Reporter / Q000235:genetics; D006801:Humans
D001057:Apolipoproteins E