Mutational analysis of the Lem3p-Dnf1p putative phospholipid-translocating P-type ATPase reveals novel regulatory roles for Lem3p and a carboxyl-terminal region of Dnf1p independent of the phospholipid-translocating activity of Dnf1p in yeast.
Lem3p-Dnf1p is a putative aminophospholipid translocase (APLT) complex that is localized to the plasma membrane; Lem3p is required for Dnf1p localization to the plasma membrane. We have identified lem3 mutations, which did not affect formation or localization of the Lem3p-Dnf1p complex, but caused a synthetic growth defect with the null mutation of CDC50, a structurally and functionally redundant homologue of LEM3. Interestingly, these lem3 mutants exhibited nearly normal levels of NBD-labeled phospholipid internalization across the plasma membrane, suggesting that Lem3p may have other functions in addition to regulation of the putative APLT activity of Dnf1p at the plasma membrane. Similarly, deletion of the COOH-terminal cytoplasmic region of Dnf1p affected neither the localization nor the APLT activity of Dnf1p at the plasma membrane, but caused a growth defect in the cdc50Delta background. Our results suggest that the Lem3p-Dnf1p complex may play a role distinct from its plasma membrane APLT activity when it substitutes for the Cdc50p-Drs2p complex, its redundant partner in the endosomal/trans-Golgi network compartments.
Noji
Takehiro
T
Division of Molecular Interaction, Institute for Genetic Medicine, Hokkaido University, Sapporo 060-0815, Japan.
Yamamoto
Takaharu
T
Saito
Koji
K
Fujimura-Kamada
Konomi
K
Kondo
Satoshi
S
Tanaka
Kazuma
K
D018528:ATP-Binding Cassette Transporters; D000251:Adenosine Triphosphatases / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D000595:Amino Acid Sequence; D001692:Biological Transport; D000252:Calcium-Transporting ATPases / Q000235:genetics / Q000378:metabolism / Q000502:physiology; D002462:Cell Membrane / Q000201:enzymology; D004252:DNA Mutational Analysis; D026901:Membrane Transport Proteins / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D008969:Molecular Sequence Data; D049294:Phospholipid Transfer Proteins / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D010743:Phospholipids / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000235:genetics; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D017384:Sequence Deletion
D018528:ATP-Binding Cassette Transporters; C427246:CDC50 protein, S cerevisiae; C104014:DRS2 protein, S cerevisiae; C478771:Lem3 protein, S cerevisiae; D026901:Membrane Transport Proteins; D049294:Phospholipid Transfer Proteins; D010743:Phospholipids; D029701:Saccharomyces cerevisiae Proteins; D000251:Adenosine Triphosphatases; D000252:Calcium-Transporting ATPases; C480601:Dnf1 protein, S cerevisiae