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First published online April 3, 2008
doi: 10.1242/10.1242/jcs.016998

Journal of Cell Science 121, 1293-1302 (2008)
Published by The Company of Biologists 2008
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Ypt1p is essential for retrograde Golgi-ER transport and for Golgi maintenance in S. cerevisiae

Faustin Kamena1,*, Melanie Diefenbacher1,2,*, Cornelia Kilchert2, Heinz Schwarz3 and Anne Spang1,2,{dagger}

1 Friedrich Miescher Laboratory of the Max Planck Society, Spemannstrasse 39, 72076 Tübingen, Germany
2 Biozentrum, University of Basel, Klingelbergstrasse 70, CH-4056 Basel, Switzerland
3 Max Planck Institute for Developmental Biology, Spemannstrasse 35, 72076 Tübingen, Germany


Figure 1
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  		Fig. 1. Ypt1p is required for retrograde transport from the Golgi to the ER. (A) Addition of GDI reduced the retrieval efficiency of [35S]gp{alpha}F-HDEL to the ER. A round-trip assay was performed with wild-type semi-intact cells. GDI was added to the round-trip assay after the fusion of COPII vesicles with the ER. The amount of trimmed [35S]gp{alpha}F-HDEL in lane 2 was reduced to 63% compared with that in the buffer control (100%). In the buffer control 1.14% and after addition of GDI 0.73% of the [35S]gp{alpha}F-HDEL incorporated into COPII vesicles at the donor ER reached the acceptor ER and was trimmed. (B) YPT mutants are not defective in retrograde transport at the permissive temperature. Semi-intact cells from different ts-mutants and wild type were used as acceptor membranes in a round-trip assay. The last step, the retrieval from the Golgi to the ER, was performed at the permissive temperature, 23°C. All semi-intact cells gave a comparable signal of trimmed [35S]gp{alpha}F-HDEL. (C) Deletion of YPT1 results in a reduction of retrograde transport from the Golgi to the ER. In parallel to the assay in B an assay was performed, in which the temperature was raised in the last step to 30°C. This temperature should represent at least a semi-restrictive temperature for most ts-mutants. The signal did not alter significantly for most mutants when compared with wild type, but the signal of retrieved and trimmed [35S]gp{alpha}F-HDEL was strongly diminished in {Delta}ypt1/SLY1-20 acceptor membranes. (D) SLY1-20 expression does not contribute to the defect in retrograde transport. A retrieval assay was performed as described above. In {Delta}ypt1/SLY1-20 membranes the signal in the retrograde transport assay was drastically reduced at 30°C, but the transport in WT/SLY1-20 was as efficient as in WT. (E) ypt1-3 is not defective in retrograde transport in vitro. A retrieval assay was performed to compare two different YPT1 mutants side by side. {Delta}ypt1/SLY1-20 membranes were unable to allow retrograde transport of [35S]gp{alpha}F-HDEL at the restrictive temperature, ypt1-3 membranes behaved like wild-type semi-intact cells. `Bckgrd' is an assay using wild-type membranes, but cytosol was omitted in the last incubation step (transport from the Golgi to the ER). The retrieval efficiency was determined as percentage of trimmed [35S]gp{alpha}F-HDEL of the reporter that was incorporated into COPII-coated vesicles at the ER. At 20°C: WT 2.9 %, {Delta}ypt1/SLY1-20 2.34 %, ypt1-3 3.15 %; at 30°C: WT 2.92 %, {Delta}ypt1/SLY1-20 1.28%, ypt1-3 3.05%.

 

Figure 2
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  		Fig. 2. Behavior of YPT mutants in anterograde transport and in a Kar2p secretion assay. (A,B) An ER-Golgi transport assay. The arrival of [35S]gp{alpha}F-HDEL in the Golgi was monitored by precipitation with {alpha}-1,6-mannose antibodies and protein A-Sepharose. The amount of gp{alpha}F-HDEL precipitated by {alpha}-1,6-mannose antibodies was normalized to the amount of budded gp{alpha}F-HDEL from the ER. The average and standard deviation from at least three independent experiments are shown. The ER-Golgi transport assay was performed at (A) 23°C and (B) 30°C. (C) Mutants in YPT1 secrete Kar2p. Colony blot of different YPT mutants grown overnight at 30°C. Secreted Kar2p was detected by immunoblotting.

 

Figure 3
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  		Fig. 3. COPI-coated vesicles from {Delta}ypt1/SLY1-20 Golgi are depleted from cargo and SNAREs. Golgi membranes enriched from either wild-type (A) or {Delta}ypt1/SLY1-20 (B) cells were incubated with Arf1p, coatomer and GTP-{gamma}-S, to generate COPI vesicles. The vesicles were separated by velocity sedimentation centrifugation. The vesicle peak was collected and floated on a buoyant density gradient. Fractions were collected from the top, precipitated, and analyzed by immunoblotting. Sec22p is a v-SNARE in the ER-Golgi shuttle and Emp47p is a vesicle cargo. The arrows indicate the movement of the lipid particles in the gradient.

 

Figure 4
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  		Fig. 4. Wild-type and {Delta}ypt1/SLY1-20 Golgi differ in their protein content. Immunoblots of equal amounts of Golgi membranes enriched from wild-type, wild-type expressing SLY1-20 and {Delta}ypt1/SLY1-20 cells that were grown at 23°C. The blots were developed with antibodies directed against the Golgi enzymes Anp1p and Mnn1p, the SNAREs Sed5p, Bos1p and Sec22p, the cargo Emp47p, and the COPI components Arf1p and coatomer. (A) A representative collection of immunoblots. (B) The immunoblots were quantified using a Licor Odyssey system. The average and standard deviation from at least three independent experiments are shown. (C) Proteins that are lost from {Delta}ypt1/SLY1-20 Golgi are not degraded. Total cell lysates were prepared from cells that were grown at 23°C or shifted to 37°C. Equal amounts of protein were loaded for SDS-PAGE and analyzed by immunoblotting with antibodies as described above and with anti-Pgk1 as a loading control.

 

Figure 5
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  		Fig. 5. The localization of various Golgi proteins is altered in {Delta}ypt1/SLY1-20 cells. Cells were grown to early log phase at 23°C. One half of the culture was shifted to 37°C for 1 hour. Cells were either examined directly (Anp1p-GFP, Sed5p-GFP, Sec7p-dsRed) or treated for immunofluorescence (Emp47p-myc) with mouse anti-myc and goat-anti mouse IgGs coupled to CY3.

 

Figure 6
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  		Fig. 6. Golgi function is impaired in {Delta}ypt1/SLY1-20 cells. (A) Glycosylation is enhanced in {Delta}ypt1/SLY1-20 cells. Strains were grown overnight at 23°C to early to mid log phase and shifted for 1 hour to 37°C where indicated. Total yeast lysates were prepared and equal amounts of protein were separated by SDS-PAGE. Glycosylated proteins were visualized with a Pro-Q Emerald 300 Kit. More glycoproteins were detected in {Delta}ypt1/SLY1-20 cells irrespective of the temperature. M, marker; pos. con., positive control (HRP); neg. con., negative control, Sec24p; GM, CandyCane glycoprotein molecular mass standards: (from top to bottom) 180 kDa; 82 kDa; 42 kDa; 18 kDa. (B) CPY outer chain glycosylation still occurs at 23°C and is abolished 37°C in {Delta}ypt1/SLY1-20 cells. A pulse-chase experiment was performed at either 23°C or 37°C. Transport of CPY to the vacuole was delayed, yet the glycosylation occurred normally at 23°C in {Delta}ypt1/SLY1-20 cells. At 37°C transport between the ER and the Golgi is blocked. Hence no CYP maturation could occur. Note that a wild-type strain expressing SLY1-20 behaved like the wild type in the assay.

 

Figure 7
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  		Fig. 7. Ultrastructural analysis of {Delta}ypt1/SLY1-20 cells. Wild-type, wild-type expressing SLY1-20 and {Delta}ypt1/SLY1-20 strains were grown to early to mid-log phase and half of the culture was shifted to 37°C for 1 hour. The cultures were processed for ultrastructural analysis. Thin sections were stained with lead citrate and uranyl acetate. (A-D) Cells grown at 23°C; (E-H) cells shifted for 1 hour to 37°C. (A,E) wild-type; (B,F) wild-type expressing SLY1-20; (C,D,G,H) {Delta}ypt1/SLY1-20. White arrowheads point to individual Golgi cisternae. The black arrowhead in G points to an accumulation of vesicles [an enlargement (x2) is shown in the inset]. The black arrowhead in H is directed towards strange membraneous profiles. An enlargement (x2) is shown in the inset. N, nucleus; V, vacuole. Bar (in A), 500 nm.

 

Figure 8
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  		Fig. 8. Ypt1p interacts directly with Ufe1p-GST. The transmembrane domain of the SNAREs was replaced by GST. Equal amounts of GST-tagged SNAREs were immobilized on GSH-agarose and incubated with purified His6-Ypt1p. His6-Ypt1p bound to the SNAREs was detected by immunoblotting (lanes 1-10). The amount of immobilized SNARE proteins is shown in lanes 11-20.

 

Figure 9
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  		Fig. 9. UFE1 and YPT1 interact genetically. (A) Schematic outline of the cross between ypt1-3 and {Delta}ufe1 pufe1-1. A subset of the meiotic outcomes is shown. (B) Determination of the transmission rate of pufe1-1 in wild-type yeast spores.

 

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© The Company of Biologists Ltd 2008