Partial repartitioning of the NST-adjoining TADs across membranes into the cyto/nucleoplasm.

Abstract

<p>(<b>A</b>) Schematic of a series of Nrf1 deletion mutants lacking discrete portions of AD1 (including Neh5L and DIDLID/DLG), TMi-containing NST, AD2, SR, TMp-containing Neh6L, and bZIP. In addition, the locations of the eN mutants are also indicated across the AD2, SR and Neh6L domains. (<b>B</b> and <b>C</b>) Cells expressing wild-type Nrf1 (<b><i>b1</i></b>), its mutant Nrf1^Δ280-298, Nrf1^Δ171-186 (<b><i>b2</i></b><b>)</b>, or others indicated (<b><i>C</i></b>) were subjected to subcellular fractionation, followed immediately by an intact ER membrane protection assay to measure the sensitivity of the ectopic proteins to digestion by PK (50 μg/ml); proteolysis was allowed to proceed in the presence or absence of 1% TX in reaction mixtures placed on ice. The products were examined by immunoblotting with polyclonal antibodies against Nrf1β before being re-probed with antibodies against calreticulin (CRT) as a marker for luminal proteins. The intensity of these blots was estimated by dividing the value for Nrf1 with that for CRT, and the relative percentage (%) amount of Nrf1 that remained after PK digestion was normalized to the total amount of Nrf1 in reactions without PK digestion. The results are shown graphically (<i>c,</i> mean ± S.D, n = 4), allowing the stability of different Nrf1 mutants in membrane PK protection reactions to be compared (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0093458#pone.0093458.s004" target="_blank">Figure S4</a>). (<b>D</b> and <b>E</b>) Membrane PK protection reactions using intact ER-enriched fractions purified from cells expressing Nrf1^Δ374-393, Nrf1^Δ409-428, Nrf1^Δ466-488, Nrf1^Δ508-513 or Nrf1^Δ519-537 proteins (<b><i>D</i></b>) or other mutants indicated (<b><i>E</i></b>). The relative percentage of protein remaining after PK digestion was calculated as described above. The results are shown graphically (<i>e,</i> mean ± S.D, n = 4), allowing the stability of different Nrf1 mutants in membrane PK protection reactions to be compared (also see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0093458#pone.0093458.s006" target="_blank">Figure S6</a>). (<b>F</b>) The <i>left</i> schematic shows Nrf1 mutants lacking various portions of the protein. Their contributions to changes in Nrf1 activity in response to glucose starvation, when compared with activity observed under 25 mM-glucose conditions (control), were examined using the reporter assay. Significant increases ($, p<0.05 and$$, p<0.001, n = 9) and decreases (*p<0.05, **p<0.001, n = 9) are indicated, relatively to the wild-type Nrf1 activity obtained from the 25 mM-glucose conditions.</p