Abstract
YIL023C encodes a member of the SLC39A, or ZIP, family, which we refer to as yeast KE4 (YKE4) after its mouse ortholog. Yke4p was localized to the endoplasmic reticulum (ER) membrane using Yke4p-specific antiserum. YKE4 is not an essential gene; however, deletion of YKE4 resulted in a sensitivity to calcofluor white and poor growth at 36 degrees C on respiratory substrates containing high zinc. Overexpression of transition metal transporters Zrc1p and Cot1p or the mouse orthologue mKe4 in Deltayke4 suppressed the poor growth at 36 degrees C on respiratory substrates. We found that the role of Yke4p depends on the zinc status of the cells. In a zinc-adequate environment, Yke4p transports zinc into the secretory pathway, and the deletion of YKE4 leads to a zinc-suppressible cell wall defect. In high zinc medium, transport of zinc into the secretory pathway through Yke4p is a way to eliminate zinc from the cytosol, and deletion of YKE4 leads to toxic zinc accumulation in the cytosol. Under low cytosolic zinc conditions, however, Yke4p removes zinc from the secretory pathway, and deletion of YKE4 partially compensates for the loss of Msc2p, an ER zinc importer, and therefore helps to alleviate ER stress. In our model, Yke4p balances zinc levels between the cytosol and the secretory pathway, whereas the previously described Msc2p-Zrg17p ER zinc importer complex functions mainly in zinc-depleted conditions to ensure a ready supply of zinc essential for ER functions, such as phospholipid biosynthesis and unfolded protein response.
Highlights
The Ke4-like proteins share many features with the LIV1 group, they form a separate subfamily that is present in all investigated eukaryotes
Since the mouse Ke4 gene can substitute for the yeast and A. thaliana genes, we suggest that bidirectional transport may be conserved in all eukaryotes
Lysates from ⌬yke4 cells transformed with a plasmid containing yeast KE4 (YKE4) expressed under the control of its endogenous promoter showed a novel band migrating at ϳ33 kDa that was not seen in lysates from ⌬yke4 cells (Fig. 1A)
Summary
YKE4 and PSD1 (phosphatidylserine decarboxylase 1) were deleted in W303 wild type cells (DY150), utilizing the KanMX4 cassette. KanMX4 cassette-disrupted open reading frames were PCR-amplified from the genome-wide deletion collection (Invitrogen) using the following primers: YKE4 PR1032, 5Ј-AGT AGA ATT TCT GCG GAG AAC-3Ј and PR1033 5Ј-TCC AGT TAT CGA ATT CTC CTG-3Ј; PSD1 PR1235, 5Ј-GG TAC GTA GCG CTT TAA GGA-3Ј; and PR1236, 5Ј-AAG GGG TAC ATG ACA TGG CT-3Ј. The PCR-amplified yke4::KanMX4 cassette was transformed into DY150 cells. Psd1::KanMX4 cassette was transformed into both DY1457 (MAT␣) and DY150 (MATa) strains. DY1457⌬psd was used to mate the DY150-based strains to create double and triple mutant cells. The double and triple deletion strains were confirmed by PCR analysis. YKE4 was cloned and expressed with its own promoter (defined as a 1000-bp-long 5Ј region of the start codon) into high copy episomal (pTF63) and low copy centromeric (YCp33) plasmids using PR1035
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