Loss of Drs2p does not abolish transfer of fluorescence-labeled phospholipids across the plasma membrane of Saccharomyces cerevisiae.
The yeast DRS2 gene, which is required for growth at 23 degreesC or below, encodes a member of a P-type ATPase subgroup reported to transport aminophospholipids between the leaflets of the plasma membrane. Here, we evaluated the potential role of Drs2p in phospholipid transport. When examined by fluorescence microscopy, a drs2 null mutant showed no defect in the uptake or distribution of fluorescent-labeled 1-palmitoyl-2[6-(7-nitrobenz-2-oxa-1, 3-diazol-4-yl (NBD))aminocaproyl]phosphatidylserine) or 1-myristoyl-2[6-NBD-aminocaproyl]phosphatidylethanolamine. Quantification of the amount of cell-associated NBD fluorescence using flow cytometry indicated a significant decrease in the absence of Drs2p, but this decrease was not restricted to the aminophospholipids (phosphatidylserine and phosphatidylethanolamine) and was dependent on culture conditions. Furthermore, the absence of Drs2p had no effect on the amount of endogenous PE exposed to the outer leaflet of the plasma membrane as detected by labeling with trinitrobenzene sulfonic acid. The steady state pool of Drs2p, which was shown to reside predominantly in the plasma membrane, increased upon shift to low temperature or exposure to various divalent cations (Mn2+, Co2+, Ni2+, and Zn2+ but not Ca2+ or Mg2+), conditions that also inhibited the growth of a drs2 null mutant. The data presented here call into question the identification of Drs2p as the exclusive or major aminophospholipid translocase in yeast plasma membranes (Tang, X., Halleck, M. S., Schlegel, R. A., and Williamson, P. (1996) Science 272, 1495-1497).
Siegmund
A
A
Institut für Biochemie der Universität Stuttgart, Pfaffenwaldring 55, D-70569 Stuttgart, Germany.
Grant
A
A
Angeletti
C
C
Malone
L
L
Nichols
J W
JW
Rudolph
H K
HK
D009327:4-Chloro-7-nitrobenzofurazan; D001483:Base Sequence; D001692:Biological Transport; D000252:Calcium-Transporting ATPases / Q000235:genetics* / Q000378:metabolism*; D002413:Cations, Divalent / Q000494:pharmacology; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism; D002874:Chromosome Mapping; D017931:DNA Primers; D005456:Fluorescent Dyes; D005656:Fungal Proteins / Q000235:genetics / Q000378:metabolism; D015966:Gene Expression Regulation, Fungal / Q000187:drug effects; D008670:Metals / Q000494:pharmacology; D008856:Microscopy, Fluorescence; D008969:Molecular Sequence Data; D010743:Phospholipids / Q000378:metabolism*; D016133:Polymerase Chain Reaction; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics* / Q000254:growth & development / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins
D002413:Cations, Divalent; D017931:DNA Primers; C104014:DRS2 protein, S cerevisiae; D005456:Fluorescent Dyes; D005656:Fungal Proteins; D008670:Metals; D010743:Phospholipids; D011994:Recombinant Proteins; D029701:Saccharomyces cerevisiae Proteins; D000252:Calcium-Transporting ATPases; D009327:4-Chloro-7-nitrobenzofurazan