An essential subfamily of Drs2p-related P-type ATPases is required for protein trafficking between Golgi complex and endosomal/vacuolar system - PubMed (original) (raw)

Zhaolin Hua et al. Mol Biol Cell. 2002 Sep.

Abstract

The Saccharomyces cerevisiae genome contains five genes encoding P-type ATPases that are potential aminophospholipid translocases (APTs): DRS2, NEO1, and three uncharacterized open reading frames that we have named DNF1, DNF2, and DNF3 for DRS2/NEO1 family. NEO1 is the only essential gene in APT family and seems to be functionally distinct from the DRS2/DNF genes. The drs2Delta dnf1Delta dnf2Delta dnf3Delta quadruple mutant is inviable, although any one member of this group can maintain viability, indicating that there is a substantial functional overlap between the encoded proteins. We have previously implicated Drs2p in clathrin function at the trans-Golgi network. In this study, we constructed strains carrying all possible viable combinations of null alleles from this group and analyzed them for defects in protein transport. The drs2Delta dnf1Delta mutant grows slowly, massively accumulates intracellular membranes, and exhibits a substantial defect in the transport of alkaline phosphatase to the vacuole. Transport of carboxypeptidase Y to the vacuole is also perturbed, but to a lesser extent. In addition, the dnf1Delta dnf2Delta dnf3Delta mutant exhibits a defect in recycling of GFP-Snc1p in the early endocytic-late secretory pathways. Drs2p and Dnf3p colocalize with the trans-Golgi network marker Kex2p, whereas Dnf1p and Dnf2p seem to localize to the plasma membrane and late exocytic or early endocytic membranes. We propose that eukaryotes express multiple APT subfamily members to facilitate protein transport in multiple pathways.

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Figures

Figure 1

Figure 1

DRS2 and DNFs constitute an essential gene family. (A) BY4742 (WT), ZHY615D1C (_drs2_Δ), BY4742 YER166W (_dnf1_Δ), ZHY2143A (_drs2_Δ _dnf1_Δ), ZHY709 (_drs2_Δ _dnf1_Δ _dnf2_Δ), ZHY708 (_drs2_Δ _dnf1_Δ _dnf3_Δ), ZHY7282C (_drs2_Δ _dnf2_Δ _dnf3_Δ), and PFY3273A (_dnf1_Δ _dnf2_Δ _dnf3_Δ) strains were grown on YPD plates at 30, 37, or 20°C as indicated in the lower right panel. (B) The _dnf1,2,3_Δ _drs2_Δ quadruple mutant is inviable. Serial dilutions of strains PFY3273A pRS416-DRS2 (_dnf1,2,3_Δ pRS416-DRS2) and ZHY704 (_dnf1,2,3_Δ _drs2_Δ pRS416-DRS2) were spotted on YPD or minimal 5-FOA plates, and incubated at 30°C (shown), and 37, 24, 20, or 15°C (not shown).

Figure 2

Figure 2

_drs2_Δ _dnf1_Δ mutant massively accumulates abnormal membrane bound structures resembling Berkeley bodies. BY4742 (WT), ZHY2149D (_drs2_Δ _dnf1_Δ), and PFY3273A (_dnf1,2,3_Δ) cells were prepared for electron microscopy as described previously (Chen et al., 1999). Numerous double-membrane ring structures resembling Berkeley bodies (indicated with arrows) accumulate in _drs2_Δ _dnf1_Δ cells, whereas the _dnf1,2,3_Δ cells were similar to wild-type cells.

Figure 3

Figure 3

Drs2p and Dnf1p are required for ALP transport to the vacuole. (A) Localization of GFP-ALP. BY4742 (WT), ZHY615D1C (_drs2_Δ), ZHY2149D (_drs2_Δ _dnf1_Δ), BY4742 YER166W (_dnf1_Δ), PFY3273A (_dnf1,2,3_Δ), and 6210 _apl5_Δ harboring pGO41 (GFP-ALP) were grown at 30°C to log phase and examined by fluorescence microscope. (B) Percentage of cells exhibiting abnormal GFP-ALP localization. Cells containing 0, 1–3, 4–6, 7–9, or ≥10 punctate structures bearing GFP-ALP were counted and presented as the percentage of the total number of cells counted. Data shown is the average from counting >100 cells from three independent transformants of each strain. (C) Steady-state distribution of ALP forms. Cells were grown in YPD media at 30°C to log phase. Whole cell lysates were prepared and immunoblotted to detect precursor (pro) and mature (m) forms of ALP. (D) Kinetics of ALP transport to the vacuole. Cells were grown to log phase at 30°C, labeled with [35S]methione/cysteine for 5 min and chased at 0, 5, 10, and 20 min at 30°C. ALP was recovered from each sample by immunoprecipitation and subjected to SDS-PAGE. (E) CPY and ALP processing rate assessed from pulse-chase experiments were plotted relative to WT cells. Data shown is the average of at least three experiments.

Figure 4

Figure 4

CPY pathway is partially affected in drs2/dnf deletion strains at 30°C. (A) Colony-blot analysis of CPY secretion. Freshly streaked BY4742 (WT), BY4742 YER166W (_dnf1_Δ), PFY3273A (_dnf1,2,3_Δ), ZHY615D1C (_drs2_Δ), ZHY2149D (_drs2_Δ _dnf1_Δ) and 6210 _vps35_Δ cells were grown on YPD plates at 30°C (shown), and 37 or 20°C (data not shown) for 24 h. Nitrocellulose membranes were overlaid onto the colonies and incubated for another 24 h. Membranes were washed and probed with a monoclonal CPY antibody. (B) Steady-state distribution of CPY precursor forms. Cells were grown in YPD medium at 30°C to log phase. Whole cell lysates were prepared and subjected to SDS-PAGE and Western blotting to detect p1, p2 and mature forms of CPY. (C) Kinetics of CPY transport. Cells were grown to log phase at 30°C, labeled with [35S]methionine/cysteine for 5 min, and chased at 0, 5, and 10 min at 30°C. CPY was recovered from each sample by immunoprecipitation and subjected to SDS-PAGE.

Figure 5

Figure 5

dnf1Δ does not exacerbate the pro-α-factor processing and invertase secretion defects of _drs2_Δ. (A) Kinetics of pro-α-factor processing. Cells were grown to log phase at 30°C, labeled with [35]methione/cysteine for 5 min, and chased at 0, 5, and 10 min at 30°C. Proα-factor was recovered from each sample by immunoprecipitation and subjected to SDS-PAGE. (B) Invertase secretion assay. Cells were grown in 5% YP glucose media at 30°C to log phase and shifted to 0.1% YP glucose media to start the induction of invertase. Aliquots of cells were collected at 0, 15, 30, and 45 min after invertase is induced. Secreted and total invertase activities were measured and expressed as the percentage of invertase secreted.

Figure 6

Figure 6

_dnf1,2,3_Δ mutant exhibits a cold-sensitive defect in FM4-64 transport to the vacuole. Cells were stained with FM4-64 on ice and shifted to 15°C for 0, 1, or 4 h and then viewed by fluorescence microscopy.

Figure 7

Figure 7

_dnf1,2,3_Δ mutant exhibits a defect in Snc1p-GFP recycling. (A) pRS416 _SNC1-_GFP was introduced into BY4742 (WT), PFY3273A (_dnf1,2,3_Δ) and ZHY615D1C (_drs2_Δ) strains. Cells were grown at 30°C to log phase and examined by fluorescence microscopy. (B) Cells harboring pRS426 STE2-GFP were grown at 30°C to log phase and examined by fluorescence microscopy. MetaMorph 4.5 was used to process the image and measure the fluorescence intensity on cell surface and in the vacuoles.

Figure 8

Figure 8

Expression levels of Drs2/Neo1 family proteins. (A) Strains BY4742 (control), ZHYNEO1-MYC, ZHYDRS2-MYC, ZHYDNF1-MYC, ZHYDNF2-MYC, and ZHYDNF3-MYC were grown in YPD media at 30°C. Equal amounts of whole cell lysates were subjected to SDS-PAGE and immunoblotting with a monoclonal c-Myc antibody to detect the fusion proteins. The migration of standard protein markers is labeled at the left. Predicted molecular mass of each full-length protein fused with 13XMyc tag is Neo1p, 146 kDa; Drs2p, 170 kDa; Dnf1p, 194 kDa; Dnf2p, 198 kDa; and Dnf3p, 204 kDa. Each lane contained an equivalent amount of CPY (not shown). (B) Full length and near full-length bands from A were analyzed by NIH Image 1.62. The signal intensity of each fusion protein was plotted as the percentage of Drs2p signal strength. Data shown in this figure is representative of three experiments.

Figure 9

Figure 9

Localization of Dnf proteins. Cells were grown in YPD media at 30°C and prepared for immunofluorescence. (A) Cells were visualized with mouse monoclonal c-Myc antibody (a–d). (B) Cells transformed with a 2 μ KEX2HA plasmid were stained with a mouse monoclonal c-Myc antibody and a rabbit polyclonal HA antibody to visualize Myc-tagged Drs2/Dnf proteins (red) and HA-tagged Kex2p (green) (e–h). The yellow punctate structures indicate the colocalization of Drs2/Dnf proteins with Kex2p.

Figure 10

Figure 10

Model for the roles of Drs2p and Dnf proteins in late secretory, vacuolar, and endocytic pathways. PM, plasma membrane; EE, early endosome; LE, late endosome.

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