Dtr1p, a Multidrug Resistance Transporter of the Major Facilitator Superfamily, Plays an Essential Role in Spore Wall Maturation in Saccharomyces cerevisiae

Abstract

The de novo formation of multilayered spore walls inside a diploid mother cell is a major landmark of sporulation in the yeast Saccharomyces cerevisiae. Synthesis of the dityrosine-rich outer spore wall takes place toward the end of this process. Bisformyl dityrosine, the major building block of the spore surface, is synthesized in a multistep process in the cytoplasm of the prospores, transported to the maturing wall, and polymerized into a highly cross-linked macromolecule on the spore surface. Here we present evidence that the sporulation-specific protein Dtr1p (encoded by YBR180w) plays an important role in spore wall synthesis by facilitating the translocation of bisformyl dityrosine through the prospore membrane. DTR1 was identified in a genome-wide screen for spore wall mutants. The null mutant accumulates unusually large amounts of bisformyl dityrosine in the cytoplasm and fails to efficiently incorporate this precursor into the spore surface. As a result, many mutant spores have aberrant surface structures. Dtr1p, a member of the poorly characterized DHA12 (drug:H ؉ antiporter with 12 predicted membrane spans) family, is localized in the prospore membrane throughout spore maturation. Transport by Dtr1p may not be restricted to its natural substrate, bisformyl dityrosine. When expressed in vegetative cells, Dtr1p renders these cells slightly more resistant against unrelated toxic compounds, such as antimalarial drugs and food-grade organic acid preservatives. Dtr1p is the first multidrug resistance protein of the major facilitator superfamily with an assigned physiological role in the yeast cell.Diploid a/␣ cells of the budding yeast Saccharomyces cerevisiae undergo a specialized developmental program termed sporulation when transferred to a nitrogen-free medium containing potassium acetate as nonfermentable carbon source. The final product of sporulation is an ascus that consists of four haploid spores surrounded by the ascus wall, the former vegetative cell wall (for a review, see reference 27). Spores are protected from adverse environmental conditions by the spore wall. Especially the surface layers contribute both to the spores' mechanical rigidity and their resistance against chemical and enzymatic attack (3). Spore wall synthesis begins with the formation of the prospore membrane, a bilayered electrondense structure that starts to form during the second meiotic division on the cytoplasmic side of each of the four spindle pole bodies by fusion of secretory vesicles (9, 13, 23, 31-33). As meiosis progresses, the prospore membrane extends along the outer surface of the nuclear envelope. Septins, among them the sporulation-specific proteins Spr3p and Spr28p, localize at the leading tip of the prospore membrane under control of the Gip1p-Glc7p phosphatase complex and might be involved in its extension and directed growth (14,16,44). Other proteins localized to the growing prospore membrane are SspIp, Ady3p, and Don1p, which form the leading edge protein coat (23,33) and presumably Sps2p (11,38), but th...

Phospholipase D and the SNARE Sso1p are necessary for vesicle fusion during sporulation in yeast

“…Therefore, it is possible that it localized to these vesicles without trafficking through the secretory pathway. To examine whether an integral membrane protein of the prospore membrane would also be found in these clusters, a fusion of GFP to the Dtr1p transporter was examined (Felder et al, 2002). In wild-type cells, Dtr1p-GFP was found exclusively on the prospore membrane, as reported previously (Felder et al, 2002).…”

“…To examine whether an integral membrane protein of the prospore membrane would also be found in these clusters, a fusion of GFP to the Dtr1p transporter was examined (Felder et al, 2002). In wild-type cells, Dtr1p-GFP was found exclusively on the prospore membrane, as reported previously (Felder et al, 2002). By contrast, in both the sso1 and spo14 cells Dtr1p-GFP was found in foci adjacent to the SPB, similar to GFP-Spo14 K1098H p. With both GFP Journal of Cell Science 119 (7) fusions, the intensity of fluorescence from the SPB region was slightly less prominent in the spo14 cells than in the sso1 cells.…”

Sporulation in the Budding Yeast Saccharomyces cerevisiae

“…The dityrosine is exported from the cytosol by the action of a dedicated MDR family transporter, Dtr1 (Felder et al 2002), and then polymerized into a much larger structure that assembles on the surface of the chitosan (Briza et al 1990b). Incorporation into the polymer leads to the isomerization of 50% of the dityrosine molecules from the L,L stereoisomer to the D,L form (Briza et al 1990b).…”

Erv14 family cargo receptors are necessary for ER exit during sporulation inSaccharomyces cerevisiae

“…The plasma membrane ATPase Pma1p, the t-SNARE Sso1p, and the dityrosine transporter Dtr1p (Serrano et al, 1986;Aalto et al, 1993;Felder et al, 2002) as well as Sma2p were expressed as GFP fusions either under the control of the TEF2 promoter in vegetative cells or under the SPO20 promoter in sporulating cells. The localization of these four proteins was examined in wild type, erv14⌬, erv15⌬ and erv14⌬ erv15⌬ diploids.…”