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doi:10.1111/j.1600-0854.2010.01079.x

Oligomeric Dop1p is Part of the EndosomalNeo1p-Ysl2p-Arl1p Membrane Remodeling Complex

Sonia Barbosa1, Dagmar Pratte1, Heinz

Schwarz2, Rudiger Pipkorn3 and Birgit

Singer-Kruger1,2,∗

1University of Stuttgart, Institute for Biochemistry,D-70569 Stuttgart, Germany2Max Planck Institute for Developmental Biology,Spemannstrasse 35, D-72076 Tubingen, Germany3DKFZ, D-69120 Heidelberg, Germany*Corresponding author: Birgit Singer-Kruger,birgit.singer-krueger@tuebingen.mpg.de

Yeast Dop1p is an essential protein that is highly

conserved in evolution and whose function is largely

unknown. Here, we provide evidence that Dop1p localizes

to endosomes and exists in a complex with two other

conserved proteins: Neo1p, a P4-ATPase and putative

flippase, and the scaffolding protein Ysl2p/Mon2p. The

latter operates during membrane budding at the tubular

endosomal network/trans-Golgi network (TEN/TGN) in

a process that includes clathrin recruitment via adaptor

proteins. Consistent with a role for Dop1p during this

process, temperature-sensitive dop1-3 cells accumulate

multivesicular, elongated tubular and ring-like structures

similar to those displayed by neo1 and ysl2 mutants.

In further agreement with the concept of Dop1p-Neo1p-

Ysl2p complex formation and co-operation, we show that

dop1-3 cells exhibit reduced levels of Neo1p and Ysl2p at

steady state. Conversely, mutations or deletions in NEO1

and YSL2 lead to a decrease in Dop1p levels. In addition

to binding to Neo1p and Ysl2p, Dop1p can form dimers

or multimers. A critical region for dimerization resides in

the C-terminus with leucine zipper-like domains. Dop1p’s

membrane association is largely mediated by its internal

region, but Ysl2p might not be crucial for membrane

recruitment.

Key words: Arl1, endosomes, flippases, membrane traffic,

P-type ATPase, TGN

Received 6 November 2009, revised and accepted

for publication 7 May 2010, uncorrected manuscript

published online 11 May 2010, published online 8 June

2010

The tubular endosomal network (TEN) and the trans-Golginetwork (TGN) play central roles in endocytic and biosyn-thetic membrane trafficking. These sorting compartmentsare characterized by similar pleiomorphic structures witha central cisternal region from which elaborate tubulesand vesicular structures branch off (1). The formation ofstructures with distinct membrane curvature likely relieson a complex interplay of many factors. How this occurs

and the molecular identity of the underlying machineryare questions that are still largely unsolved. Yet, a num-ber of general mechanisms seem to be used throughoutthe cell to create changes in membrane shape. Vari-ous types of proteins that are able to bend membraneshave been identified. Some of the proteins that arethought to play a significant role are protein scaffolds thatexpose a curved interaction surface to the lipid bilayer,proteins that insert amphipathic moieties into one lipidmonolayer and proteins that change the distribution ofmembrane-shaping lipids causing membrane curvaturewhen asymmetrically distributed (2,3). In particular, thequestion of how the asymmetric lipid distribution is gener-ated has been a challenging subject. Candidates for puta-tive flippases that translocate aminophospholipids suchas phosphatidylethanolamine (PE) and phosphatidylser-ine (PS) from the extracytoplasmic to the cytoplasmicleaflet are P4-ATPases, a subfamily of the large P-typeATPase superfamily (4). The first P4-ATPase identified asbeing a potential flippase was ATPase II/Atp8a1, origi-nally purified from chromaffin granules (5). The purifiedprotein was later shown to display an ATPase activityselectively and stereospecifically stimulated by PS (6).Further, using an assay based on whole yeast cells,deletion of the plasma membrane-localized Dnf1p andDnf2p was found to disrupt inward translocation of fluores-cent PS, PE and phosphatidylcholine (PC) analogues (7).Importantly, Zhou and Graham (8) recently succeeded todirectly show phospholipid translocase activity with puri-fied Drs2p reconstituted into proteoliposomes, an activityearlier identified with isolated TGN membranes and atemperature-sensitive drs2 allele (9).

The concept of transbilayer lipid translocation by flippasesbeing tightly coupled to membrane bending and subse-quent vesiculation was originally proposed based on theobservation that the addition of exogenous aminophos-pholipids to living cells results in their translocation fromthe outer to the inner membrane leaflet, followed byenhanced endocytic vesicle formation (10). Indeed, P4-ATPases are core elements of the budding machinery.In particular, yeast Drs2p and Neo1p are implicatedin clathrin-mediated pathways. The TGN-localized Drs2pexhibits genetic interactions with clathrin heavy chain,Chc1p, and the small GTPase Arf1p (11), and it phys-ically interacts with Gea2p, a GBF family member ofthe large Sec7 Arf GTP exchange factors (GEFs) (12).Loss of Drs2p causes impaired formation of a spe-cific class of clathrin-coated vesicles (11,13). Neo1p,localizing primarily to endosomes, displays genetic andbiochemical interactions with Ysl2p/Mon2p (14), a scaf-folding protein with homology to the BIG and GBFsubgroups of Sec7 GEFs (15–18). Moreover, the Arf-like

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Dop1p is Tightly Linked to Neo1p and Ysl2p

Arl1p exhibits genetic and biochemical links to Ysl2p andNeo1p (14,15,19). Expression of temperature-sensitiveneo1 alleles causes trafficking defects within the endoso-mal and vacuolar system and leads to severe alterationsof endosomal and vacuolar structures (14). Notably, theco-operation amongst Neo1p, Ysl2p and Arl1p allows theproper recruitment of the monomeric GGA clathrin adap-tors via direct interaction with Ysl2p, a process found to behighly conserved from yeast to humans (19). Collectively,this body of evidence strongly points to the participationof the P4-ATPases Drs2p and Neo1p and their specificinteraction partners in reactions that control budding atthe TGN and TEN, respectively.

Although most P-type ATPases function as a single sub-unit, several so-called β subunits of P4-ATPase wereidentified throughout eukaryotes with putative functions infolding, trafficking and enzymatic activity of its respectiveATPase subunit (20). These proteins belong to the Cdc50pfamily with three members in yeast, Ccd50p, Lem3p andCrf1p. For Drs2p, the interaction with Cdc50p appearsto be required for exit out of the endoplasmic reticulum(ER), Lem3p chaperones both Dnf1p/Dnf2p, and Crf1p isa transport chaperone of Dnf3p (21,22). As the concept ofinteraction between P4-ATPase and Cdc50p family mem-ber appeared widespread, in this study we reinvestigatedthe possibility of complex formation between Neo1p andCdc50p. While no evidence for such links was obtained,we identified the essential and highly conserved Dop1pas a new binding partner of Neo1p and its associatednetwork and showed a critical impact on the stability ofNeo1p and the associated complex subunits. The charac-terization of Dop1p domains suggests that its C-terminusis implicated in dimer/multimer formation, while its centralregion mediates the association with endosomes.

Results

Neo1p likely functions in the absence of Cdc50p

In a genetic screen, NEO1 was isolated as a multicopysuppressor of the cold-sensitive �cdc50 mutant. In sub-sequent attempts to detect physical interactions betweenCdc50p and P4-ATPases, an interaction with Drs2p butnot to Neo1p was found (21). To reinforce these studieswith our distinct experimental tools, similar experimentswere performed. However, after immunoprecipitation ofthe N-terminally tagged hemagglutinin (HA)-Neo1p versiondescribed in Ref. (14), Cdc50p-Myc was not detectableand vice versa (Figure 1A). HA-Neo1p and Ysl2p-TAP werepreviously found to physically interact in vivo (14). Giventhat a low concentration (0.01%) of the detergent Non-idet P-40 (NP-40) was critical to detect this interaction, weused similar conditions. In these conditions, approximately29% of Neo1p and 45% of Cdc50p were membrane sol-ubilized and present in a 100 000 × g supernatant, butimmunoprecipitation of either HA-Neo1p or Cdc50p-Mycdid not result in coprecipitation of the other protein (datanot shown), indicating that Neo1p does not interact with

Figure 1: Neo1p and Cdc50p do not display biochemical

and functional links. A) Immunoprecipitations (IPs) wereperformed with detergent-solubilized membranes of cells(SB90, SB121, BS1488) expressing the indicated epitope-taggedproteins using either α-Myc (lanes 1 and 2) or α-HA antibodies(lanes 3 and 4). Solubilized membrane samples and IPs wereanalyzed by immunoblotting. B) Wild-type (BS1488) and �cdc50cells (SB71) expressing HA-Neo1p were stained by indirectimmunofluorescence. Bar, 5 μm.

Cdc50p. We also compared the subcellular localization ofHA-Neo1p in wild-type and isogenic �cdc50 cells by indi-rect immunofluorescence. In contrast to non-functionalC-terminally modified Neo1p versions that are mislocal-ized to the ER (14), HA-Neo1p still localized to TEN/TGNstructures upon loss of Cdc50p, similar to cells in whichCdc50p was present (Figure 1B). Thus, the correct localiza-tion of HA-Neo1p in �cdc50 cells strongly argues againsta putative role of Cdc50p as a transport chaperone forNeo1p. We cannot exclude that Lem3p and/or Crf1p co-chaperone with Neo1p. However, as the deletion of LEM3or CRF1 causes no growth defect, in contrast to lethalitycaused by NEO1 deletion, this is highly unlikely.

Dop1p binds to Neo1p, even when Ysl2p is absent

Recent work led to the identification of Dop1p as aninteraction partner of Ysl2p. A genetic screen identi-fied DOP1 as a high copy number suppressor of the�ysl2 mutant (16) and biochemical studies showed a

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physical interaction between Dop1p and Ysl2p (16,17).This prompted us to examine in further detail the interac-tions of Dop1p with components of the Ysl2p-Arl1p-Neo1pnetwork. We constructed and expressed various epitope-tagged Dop1p versions like N-terminally tagged greenfluorescent protein (GFP)-Dop1p, expressed under theADH1 promoter from a single-copy plasmid in �dop1cells, as well as C-terminally tagged Dop1p-TAP expressedfrom the chromosomal locus. These fusions were fullyfunctional, because they complemented the deletion ofDOP1, an otherwise lethal event, and did not causelow- or high-temperature sensitivity. When GFP-Dop1pwas coexpressed with chromosomally encoded Ysl2p-HAand immunoprecipitated from detergent-solubilized cellextracts, Ysl2p-HA was coisolated. This coimmunopre-cipitation was specific, as in cells lacking GFP-Dop1pno Ysl2p-HA signal was obtained (Figure 2A). A simi-lar copurification of endogenous, non-tagged Ysl2p wasfound when Dop1p-TAP was isolated via immunoglobulin

G (IgG)-Sepharose and released from the matrix by thesite-specific tobacco etch virus (TEV) protease (data notshown).

To further establish whether Dop1p also interactswith Neo1p, Dop1p-TAP was coexpressed with HA-Neo1p. Dop1p-TAP assemblies were isolated from cellularextracts before and after a 100 000 × g centrifugationusing various NP-40 concentrations during the affinitypurification. A coisolation of HA-Neo1p with Dop1p-TAPwas indeed most successful when NP-40 was present at0.01% (Figure 2B, lane 6). Under these conditions, approx-imately 25% of Dop1p-TAP and 20% of HA-Neo1p weresolubilized as determined by centrifugation at 100 000 ×g. Importantly, isolation of detergent-solubilized Dop1p-TAP from 100 000 × g supernatants led to coisolation ofHA-Neo1p (Figure 2C). In contrast, neither the flippaseDrs2p (Figure 2C) and the endosomal t-SNARE Pep12p(data not shown), two integral membrane proteins, nor

Figure 2: Dop1p interacts with

Neo1p in vivo, independently of

Ysl2p. A) Cell extracts (BS1121,transformed with pRS315 or pRS315-PADH1-GFP-DOP1) were prepared inthe presence of 0.5% NP-40. Aftercentrifugation at 13 000 × g, super-natants (input) were subjected toimmunoprecipitations (IPs) using ananti-GFP antibody. Proteins boundto protein A–Sepharose were ana-lyzed by immunoblotting. B) Affinitypurifications were performed withcell extracts (BS862, SB205) in theabsence (lanes 1 and 5) or presenceof NP-40 (lanes 2–4 and 6–8, concen-trations as indicated). Proteins boundto IgG-Sepharose were released withthe TEV protease and detectedby immunoblotting. C) Cell extracts(BS862, SB205) were obtained in thepresence of 0.01% NP-40. For coiso-lations, 100 000 × g supernatants(input) were incubated with IgG-Sepharose (IP) and further analyzedas described in (B). D) Affinity isola-tions were performed as describedin (B) with �ysl2 (SB306, SB296)and YSL2 (SB205) cell extracts inthe absence or presence of 0.01%NP-40. E) Affinity purifications wereperformed with 100 000 × g super-natants of �ysl2 (SB306, SB296) cellextracts as described in (C).

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the clathrin heavy chain (Chc1p) (Figure 2C), which periph-erally associates with endomembranes, specifically copu-rified. Together, this shows that the interaction betweenDop1p-TAP and HA-Neo1p is highly specific and not medi-ated through membranes.

Given the Ysl2p–Dop1p interaction, we assessedwhether the Dop1p-TAP-HA-Neo1p interaction is depen-dent on Ysl2p. A �ysl2 strain expressing Dop1p-TAP andHA-Neo1p was constructed. Significantly, precipitation ofDop1p-TAP from �ysl2 cell extracts prepared with andwithout 100 000 × g centrifugation still resulted in copre-cipitation of HA-Neo1p (Figure 2D,E). The HA-Neo1p sig-nal was similar or slightly increased when compared withthat obtained with isogenic YSL2 samples (Figure 2D).Thus, Dop1p physically interacts with Neo1p in intact cellseven when Ysl2p is absent.

Mutual dependency of Dop1p and Neo1p

Given that Dop1p and Neo1p are essential proteins thatphysically interact, we further explored the links between

them by studying temperature-sensitive neo1 and dop1mutants. First, we examined two previously describedneo1 alleles, neo1-37 and neo1-69. At permissive tem-perature, HA-Neo1-69p is stable and associates withendosomes. In contrast, HA-Neo1-37p is rapidly degradedand is predominantly retained within the ER (14). Here,we tested the effects of the neo1-37 and neo1-69 alleleson Dop1p in strains in which the chromosome-encodedDop1p was N-terminally fused to GFP (GFP-Dop1p).Surprisingly, the neo1-69 mutant displayed significantlyreduced steady-state levels of GFP-Dop1p. At the permis-sive temperature, the levels of GFP-Dop1p were alreadyreduced in neo1-69 mutant cells when compared with iso-genic wild-type cells and they further decreased after shift-ing cells to 37◦C (Figure 3A). Although neo1-37 cells arehighly temperature-sensitive (Figure 3C), the GFP-Dop1plevels were not affected at 25◦C and 37◦C. For quan-titations, GFP-Dop1p was immunoprecipitated from cellextracts at the various time-points (Figure 3B). At 25◦C,only 53% of GFP-Dop1p was present in neo1-69 cells asopposed to 100 and 109% in wild-type and neo1-37 cells,

Figure 3: The neo1-69 mutant contains reduced Dop1p steady-state levels and can be suppressed by DOP1 overexpression.

A) Wild-type (SB284), neo1-69 (SB289) and neo1-37 (SB286) cells were grown at 25◦C and shifted to 37◦C for the indicated times.Cell lysates were analyzed by immunoblotting. B) GFP-Dop1p was immunoprecipitated from cell extracts obtained as described in (A).GFP-Dop1p levels were quantified by immunoblotting using the Odyssey Infrared Imaging System; the values represent averages ofthree independent experiments. C) Mutant neo1-69 (BS917) or neo1-37 (BS915) cells were transformed with the indicated plasmids andincubated as serial dilutions on yeast peptone dextrose (YPD) plates in the absence or presence of neomycin as indicated. D) GFP-Dop1pinteracts with HA-Neo1-69p, but its interaction with HA-Neo1-37p is severely reduced. Cell lysates containing either HA-Neo1p (wt)(SB201, transformed with pRS315 or pRS315-PADH1-GFP-DOP1), HA-Neo1-69p (SB463, transformed with pRS315-PADH1-GFP-DOP1) orHA-Neo1-37p (SB465, transformed with pRS315-PADH1-GFP-DOP1) were incubated with 0.01% NP-40 and subjected to centrifugationat 100 000 × g. The supernatants (input) were subjected to immunoprecipitations (IPs) using an α-GFP antibody. Proteins bound toprotein A–Sepharose were analyzed by immunoblotting.

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Figure 4: The dop1-3 mutant

exhibits reduced Neo1p and

Ysl2p steady-state levels and

can be suppressed by NEO1

overexpression. A) Schematic rep-resentation of the domain org-anization of Dop1p and location ofamino acid changes (*) in mutantDop1-3p as described in the Mate-rials and Methods. B) The dop1-3mutant (BS1806) was transformedwith the indicated plasmids and incu-bated as serial dilutions on yeastpeptone dextrose (YPD) plates in theabsence or presence of neomycin asindicated. C) Wild-type (SB323) anddop1-3 (SB142) cells were grownat 25◦C and shifted to 37◦C forthe indicated times. Cell extractswere analyzed by immunoblotting.D) Quantitation of immunoreactivebands obtained as described in (C)was performed with the OdysseyInfrared Imaging System. The val-ues are averages of five indepen-dent experiments. Values obtainedfrom the time-point 0 min werenormalized to 100%. Error bars,standard deviations. E) Cell lysateswere analyzed by immunoblotting asdescribed in (C).

respectively. After a 1-h shift to 37◦C, we observed a fur-ther reduction of the signal to 30% as compared with 81and 73% in wild-type and neo1-37 cells, respectively.These results possibly explain why the temperature-sensitive growth of neo1-69, but not neo1-37 cells, can besuppressed by DOP1 overexpression (Figure 3C). Inter-estingly, the striking and allele-specific hypersensitivityof the neo1-69 mutant to neomycin (14) was also wellsuppressed by DOP1 overexpression (Figure 3C).

We also tested whether Neo1-37p and Neo1-69p wouldstill interact with Dop1p. For this �neo1 strains weregenerated that express HA-tagged Neo1-37p or HA-Neo1-69p and GFP-Dop1p from plasmids. When GFP-Dop1pwas immunoprecipitated from 100 000 × g supernatants,wild-type HA-Neo1p and the mutant HA-Neo1-69p effi-ciently coprecipitated (Figure 3D). In contrast, coprecip-itation of HA-Neo1-37p with GFP-Dop1p was stronglyreduced to levels close to background (Figure 3D). It isimportant to state that the slightly elevated levels of

GFP-Dop1p caused by its expression from the ADH1promoter led to suppression of the neo1-69 mutant, per-haps thereby explaining the somewhat enhanced interac-tion with Dop1p. Collectively, our genetic and biochemicaldata strongly support the idea that Dop1p and Neo1pphysically co-operate in an allele-specific manner.

A mutant strain carrying the temperature-sensitive dop1-3allele with two amino acid changes in the N-terminus andfive within the C-terminal region (Figure 4A) was examinedin a similar way regarding Neo1p steady-state levels.The strain used for these studies expressed HA-Neo1pfrom the chromosome and the mutant Dop1-3p from asingle-copy plasmid in the �dop1 background. The dop1-3mutant cells grow at 25◦C but not at 37◦C (Figure 4B).As shown in Figure 4C, upon shift to 37◦C dop1-3cells displayed a gradual loss of HA-Neo1p. Conversely,two other membrane proteins, the plasma membrane-localized proton pump Pma1p, another P-type ATPase(Figure 4C), and the type I membrane protein Tlg1p (data

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not shown) were not affected, similar to phosphoglyceratekinase (PGK) that served as a loading control (Figure 4C).Thus, the specific loss of HA-Neo1p is not simply becauseof impaired membrane transport within the secretorypathway. Quantitative immunoblotting showed that aftera 3-h shift to 37◦C, the steady-state levels of HA-Neo1pwere reduced to less than 20% of those before thetemperature shift (Figure 4D). As pulse-chase analysisrevealed that in dop1-3 cells the half-life time of HA-Neo1p(synthesized during 30 min at 25◦C) was strongly reducedwhen compared with wild-type cells (t1/2 100 versus193 min; Figure S1), we can conclude that this loss ismainly because of destabilization of HA-Neo1p.

Consistent with the result that Neo1p and Dop1p aremutually affected in some dop1 and neo1 mutants, indop1-3 cells the steady-state levels of endogenous Ysl2pwere also found to be largely reduced after incubation at37◦C in contrast to isogenic DOP1 cells (Figure 4E).

Similar to neo1-69 cells, dop1-3 cells displayed hypersen-sitivity to neomycin (Figure 4B). The drug and temperaturesensitivities were both suppressed by NEO1 overexpres-sion. As overexpression of the homologous DRS2 genewas ineffective, this indicates that the suppression ofthe dop1-3 mutant by NEO1 is highly specific (Figure 4B).Overexpression of ARL1, but not YSL2, marginally rescuedthe neomycin sensitivity but not the temperature sensitiv-ity of dop1-3 cells (Figure 4B). Finally, whereas NEO1 over-expression suppressed the dop1-3 mutant (Figure 4B),this was not sufficient to rescue �dop1 cells (Figure S2A).

�ysl2 cells have reduced levels of HA-Neo1p and

Dop1p-GFP and can be rescued by overexpression

of Dop1p and Ysl2p-Arl1p-Neo1p network members

Owing to the striking interdependence of Dop1p andNeo1p, we also determined the steady-state levels ofNeo1p and Dop1p in �ysl2 cells. This mutant is viable buthighly impaired in growth at all tested temperatures, in par-ticular at 37◦C (15). In fact, �ysl2 cells displayed a dramaticreduction of HA-Neo1p levels (Figure 5A). Quantitativeanalysis revealed a decrease in HA-Neo1p to approxi-mately 32% of the levels present after complementationwith YSL2 (Figure 5A). Remarkably, overexpression ofARL1, DOP1 or NEO1 in �ysl2 cells caused a full restora-tion of HA-Neo1p levels (Figure 5A). GFP-Dop1p was alsosignificantly reduced in �ysl2 cells and the levels couldbe normalized by the extragenic suppressors ARL1, DOP1and NEO1 to an extent comparable to that achieved byYSL2 (Figure 5B). In contrast, overexpression of DRS2 didnot cause any rescue (Figure 5B). Altogether, these resultshighlight a shared interdependence of Neo1p, Dop1p andArl1p from Ysl2p (Figure 5) and they provide a plausibleexplanation for the suppression of �ysl2 cells by thesethree genes (14,16, this study).

GFP-Dop1p is present on endosomes

As in a previous study the localization of Dop1p wasreported to overlap with the TGN (17), we also addressed

Figure 5: The �ysl2 mutant displays reduced steady-state

levels of Neo1p and Dop1p and overexpression of Dop1p,

Ysl2p, Arl1p and Neo1p can rescue this loss. A) Diploid�ysl2 cells expressing HA-Neo1p (SB340) were transformedwith the indicated plasmids. Cell lysates were analyzed byquantitative immunoblotting. The values were averaged fromthree experiments and were plotted as percentage withYSL2 transformants representing 100%. B) Diploid �ysl2 cellsexpressing GFP-Dop1p (SB434) were transformed with theindicated plasmids and analyzed as described in (A).

the possibility that Dop1p localizes to endosomes. Toexamine this, GFP-Dop1p was expressed from a single-copy plasmid in �dop1 cells and observed in living cellsduring early log phase. The staining pattern was composedof numerous small dotted structures evenly distributedwithin the cell (Figure 6A). A comparable staining wasobtained in a wild-type DOP1 background (Figure 6A),suggesting that in the presence of endogenous Dop1pthe GFP-tagged fusion is not mislocalized. To examinewhether GFP-Dop1p localizes to endosomes, severalapproaches were chosen. Ideal markers for endosomes

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Figure 6: Dop1p localizes to endosomes. A) GFP-Dop1p wasexpressed from a CEN-based vector in �dop1 or DOP1 cells(SB375, BS64) and observed by fluorescence microscopy.B) BS64 cells expressing GFP-Dop1p as described in (A) wereallowed to internalize FM4-64 at 15◦C for 30 min. C) Alexa594-α-factor internalization in �ypt51 cells (BS25) transformed withCEN-based GFP-Dop1p was allowed for 40 min at 30◦C. D) GFP-Dop1p was detected in YSL2 (RH1201) and �ysl2 (BS747)transformants carrying pRS315-PADH1-GFP-DOP1. Bars (A–C),5 μm; (D), 10 μm.

are fluorescent molecules that are introduced to thecell externally and then taken up under conditionsthat allow their detection in these compartments. Thelipophilic dye FM4-64, a well-established marker for bulk-phase endocytosis, was bound at 0◦C, washed andsubsequently internalized at 15◦C to allow its detectionin early and late endosomes (23). As shown in Figure 6B,FM4-64 fluorescence frequently overlapped with GFP-Dop1p fluorescence. The only fluorescent cargo forreceptor-mediated endocytosis known in yeast is Alexadye-conjugated pheromone α-factor, recently describedby Drubin et al. (24). If Alexa594-α-factor was internalizedat 15◦C for 30 min the fluorescence was too weak fordetection, most likely because of its lack of accumulationwithin the endosomal system. Therefore, the labeledpheromone was internalized in �ypt51 cells. Becauseof blocked endocytic transport and accumulation withinearly endosomes (25,26), we were thus able to detectit. In fact, imaging of Alexa594-α-factor and GFP-Dop1pfrequently revealed an extensive degree of colocalization(Figure 6B). Further evidence for a localization of GFP-Dop1p to endosomes in wild-type cells was its vastoverlap with the endosomal protein Hse1p-mCherry (27)(Figure S3A). Finally, the expression of GFP-Dop1p in aclass E vps mutant, vps27, in which endosomes form theaberrant class E compartment and endosomal proteinscollapse to form one or few large structures (14,15),indeed revealed collapsing of GFP-Dop1p (Figure S3B)with approximately 31% of vps27 cells displaying one tothree large GFP-Dop1p structures (versus 2% in VPS27cells; n > 200, respectively). Consistent with previousevidence for colocalization of Dop1p with two proteinsthat also reside in the TGN, Arl1p and Sys1p (17), andpartial colocalization of Ysl2p/Mon2p with the late Golgi-localized GEF Sec7p (16), we also observed a partialoverlap of GFP-Dop1p and Sec7p-mCherry staining, butthe general staining patterns of both proteins weresurprisingly distinct (Figure S3C). In conclusion, our dataclearly show that within the endomembrane systemDop1p extensively localizes to structures accessibleto tracers taken up by endocytosis and to structurescontaining Hse1p. While some localization to the late Golgicould also be detected, further studies will be requiredto resolve its precise localization in the late Golgi/TGNsystem.

Deletion of CDC50 did not affect the subcellulardistribution of GFP-Dop1p (data not shown), consistentwith our observations that Cdc50p is not critical for Neo1plocalization (Figure 1B). In contrast, loss of Ysl2p clearlyreduced the signal intensity of the punctate GFP-Dop1pstructures (Figure 6D). This decrease is in agreement withthe reduced steady-state levels of GFP-Dop1p in �ysl2cells (Figure 5B). In contrast to a study in which thedeletion of YSL2 was found to cause a diffuse and possiblycytoplasmic GFP-Dop1 distribution (17), we did not detectsuch an effect on GFP-Dop1p in �ysl2 cells, as the typicalpunctate pattern was still prevalent (Figure 6D). Togetherwith the finding that the physical interaction between

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Dop1p and Neo1p is Ysl2p-independent, these resultsraise the possibility that Ysl2p does not play the major rolein membrane recruitment of Dop1p.

dop1-3 cells accumulate abnormal membrane

structures

We used electron microscopy to examine the intracellularmembranes of dop1-3 cells. When cryoimmobilized aftergrowth in the permissive conditions, a significant frac-tion of cells accumulated elongated tubular, multivesicularand ring-like structures with an increased electron density(Figure 7E,F). A typical feature of these structures wasan increased surface-to-volume ratio. After shift to thenon-permissive conditions, accumulation of the describedaberrant membrane-bound structures (Figure 7B–D) wasobserved in the majority of cells, whereas in wild-typecells similar elongated tubular and ring-like structureswere never observed (Figure 7A). As previously describedfor neo1-69 and �ysl2 mutants (14,16), in temperature-sensitive ysl2-316 cells (15) incubated for a short periodat the non-permissive conditions, we also found promi-nent morphological alterations like flattened membraneprocesses and multivesicular and ring-like structures(Figure 7G–I). Collectively, the extensive alterations inthe dop1-3 mutant hint at an important role of Dop1p inmembrane transformation events within the endomem-brane system. Moreover, the similar phenotypic changesdisplayed by mutant dop1, neo1 and ysl2 cells (thisstudy, 14,16) strongly suggest related functions of thethree interacting wild-type proteins at the TEN and TGNmembranes.

Dop1p domains are involved in manifold interactions

and contribute to distinct subcellular localizations

Dop1p is well conserved among eukaryotes, includingHomo sapiens, with a predicted molecular weight of195 kD. Sequence comparisons between orthologuesrevealed the highest degree of sequence similaritywithin the N-terminal region (Figure 4A). Another well-conserved region in the C-terminus of Dop1p orthologuesencompasses leucine zipper-like repeats with leucineor other accepted hydrophobic residues (Figure 4A). InDop1p, the three putative leucine zippers are positionedbetween residues 1502 and 1596. To examine the rolesof the distinct domains of Dop1p, the N-terminal, theinternal and the C-terminal region were fused to GFPand expressed via centromer (CEN)-based LEU2 vectorsas described for full-length Dop1p. After transformationinto �dop1 cells carrying a URA3-based DOP1 vectorand following plasmid shuffling, none of the three GFP-Dop1 fragments rescued the lethality of �dop1 cells(Figure S2B), suggesting that each of the three Dop1fragments is not functional on its own.

Next, the different GFP-Dop1 constructs were trans-formed into DOP1 cells to address their subcellularlocalization in living cells. Similar to full-length Dop1p(Figure 8A), the internal Dop1 fragment mainly localized

Figure 7: The dop1-3 mutant accumulates morphologically

abnormal membrane structures. (A–I) Wild-type (SB330) (A),dop1-3 (BS1361) (B–F) and ysl2-316 (BS1021) (G–I) cells weregrown at 25◦C and were either directly cryoimmobilized andprocessed for electron microscopy (EM) (E and F) or shiftedto 37◦C for 30 (G and H), 60 (D) or 120 (A–C, I) min, quick-frozen, and processed for EM analysis. Thin sections were viewedwith an electron microscope. M, multivesicular structure; *, ring-like membrane structure; T, elongated membrane process; bars(A and B), 1 μm; (C–I), 0.5 μm.

to several discrete puncta, likely representing TEN/TGNstructures. A significant fraction of cells (approximately28%, n > 200) displayed very bright and enlargedstructures that occasionally appeared as one or twohuge dot-like entities (Figure 8B,C). Double staining withAlexa594-α-factor or FM4-64 (Figure 8C) showed that theselarge structures overlapped with both endocytic markersand therefore correspond to endosomes. Cells expressingthe N-terminal domain displayed an enhanced cytoplas-mic fluorescence but with many faint foci remaining(Figure 8D). As this fusion was expressed at normal levels,the N-terminal domain appears to contribute only weaklyto membrane association. A very surprising, unexpectedstaining pattern was achieved with GFP-Dop1-Cterm. Inaddition to a diffuse cytoplasmic distribution, this fusionprotein accumulated in a single cellular compartment. Dou-ble staining with the DNA dye Hoechst-33342 identifiedthis site as being the nucleus (Figure 8E).

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Figure 8: Subcellular localizations and interactions of Dop1p domains. A–C) GFP-Dop1p (A) and GFP-Dop1-internal (B and C),expressed from CEN-based vectors in BS64, were observed by fluorescence microscopy. In (C), endosomes were stained withAlexa594-α-factor or FM4-64 as described in the Materials and Methods. D and E) GFP-Dop1Nterm (D) and GFP-Dop1Cterm (E) wereobserved as described in (A). In (E), nuclei were stained with Hoechst-33342. Bar, 5 μm.

We also explored the binding of the GFP-Dop1 fragmentsto Ysl2p and Neo1p by coimmunoprecipitation to narrowdown the regions within the large Dop1p implicated inthese interactions. The Dop1 fragments were expressedfrom CEN plasmids in a DOP1 background with eitherYsl2p-HA or HA-Neo1p present. Ysl2p-HA interacted morestrongly with full-length GFP-Dop1p, but binding to theC-terminal and internal fragments was still observed.Almost no interaction was seen with the N-terminus ofDop1p (Figure 9A).

Immunoprecipitation of full-length GFP-Dop1p or thethree fragments from 100 000 × g supernatants aftersolubilization with 0.01% NP-40 led to coprecipitation ofcomparable amounts of HA-Neo1p, with slightly reducedlevels for the Dop1p C-terminus (Figure 9B). Intriguingly,

an otherwise not prominent band of HA-Neo1p ofslightly increased electrophoretic mobility was enrichedafter immunoprecipitation with the internal fragment(Figure 9B, lane 4), so that altogether HA-Neo1p appearedto interact best with this Dop1p region. The identity ofthe altered or modified HA-Neo1p version is presentlyunknown.

Because of the presence of the three leucine zipper-like repeats in the C-terminal Dop1 fragment, we askedwhether this region can interact with the endogenousfull-length Dop1p. This would suggest that the regionencompassing the leucine zipper-like domains can adopt acoiled-coil structure. For these experiments, the full-lengthGFP-Dop1p or GFP-Dop1 fragments were coexpressedin a strain encoding Dop1p-HA from the chromosomal

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Figure 9: Interaction of Dop1p and

Dop1 domains with Ysl2p, Neo1p and

Dop1p. A) Cells expressing Ysl2p-HA(BS1121) and the indicated GFP-Dop1constructs from CEN-based vectorswere lysed in the presence of 0.5%NP-40. Supernatants obtained after cen-trifugation at 13 000 × g were sub-jected to immunoprecipitations usingan anti-GFP antibody. Isolated proteinswere analyzed by immunoblotting;*, IgG heavy chain. B) �neo1 cellsexpressing HA-Neo1p (SB201) and theindicated GFP-Dop1 constructs fromCEN-based vectors were lysed in thepresence of 0.01% NP-40. Super-natants obtained after centrifugationat 100 000 × g were subjected toimmunoprecipitations and analyzed asdescribed in (A); *, IgG heavy chain.C and D) Cells expressing full-lengthGFP-Dop1p (C) or the indicated GFP-Dop1 constructs (C and D) in strainsencoding Dop1p-HA (SB170) or non-tagged Dop1p (BS64) were lysed inthe presence of 0.5% NP-40 and sub-jected to immunoprecipitation using ananti-HA antibody. Isolated proteins wereanalyzed as described in (A). E) GST-Cterm-Dop1 or GST-Ypt7 was immo-bilized onto GST-Sepharose and equalquantities of His-Cterm Dop1 wereadded. Bound proteins were sepa-rated by SDS–PAGE and visualizedby Coomassie staining or immunoblot-ting. In lanes 3–6, serial dilutions (rep-resenting 3.1, 6.3, 12.5 and 25%)of the sample in lane 2 (100%) arepresent. F) Schematic summary ofthe results obtained with Dop1p sub-domains; +++, ++, + and – are thesuggested relative levels of interactions.

locus. In fact, the interaction of full-length GFP-Dop1p withfull-length Dop1p-HA (Figure 9C, lane 2) and GFP-Dop1pwith Dop1p-TAP (data not shown) was easily detectable.Next, comparing the ability of the three Dop1 fragmentsto interact with full-length Dop1p-HA showed that theC-terminal region revealed the strongest interaction, whileboth the N-terminal and the internal Dop1 region exhibitedweaker interactions (Figure 9D). The interactions of thethree fragments, even the C-terminal one, were weakerwhen compared with full-length Dop1p but clearly specific

(Figure 9C,D). We also purified the C-terminal domainof Dop1p from bacteria as recombinant glutathioneS-transferase (GST) and polyhistidine (His) fusion proteinsand performed GST pull-down experiments. Indeed,His-Dop1-Cterm bound directly to GST-Dop1-Cterm butnot to GST-Ypt7 (Figure 9E), showing that the C-terminusis able to form dimers or multimers. Quantitativeanalysis suggested that approximately 10% of the addedHis-Dop1-Cterm bound to GST-Dop1-Cterm. A summaryof the identified basic characteristics of the analyzed

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Dop1p regions regarding subcellular localizations andprotein–protein interactions is provided in Figure 9F.

Discussion

Dop1p forms a complex with Neo1p and Ysl2p

In the current study, we have identified the essentialprotein Dop1p as a crucial component of the Neo1p-Ysl2p-Arl1p network. Genetic evidence is based on thefindings that the neo1-69 and dop1-3 mutant alleles canbe suppressed by overexpression of DOP1 and NEO1,respectively. Furthermore, �ysl2 cells, previously foundto be suppressed by ARL1 and NEO1 (15,16), are alsosuppressed by DOP1. Biochemical data suggest that invivo Dop1p interacts with both Ysl2p and Neo1p. In furthersupport for complex formation and co-operation betweenDop1p, Ysl2p and Neo1p are our findings regarding thespecific loss of subcomponents in mutant dop1, neo1and ysl2 strains. Inactivation, destabilization or loss ofone subunit leads to significantly reduced amounts of theinteraction partners. Conversely, we show that the above-described extragenic suppression of �ysl2 cells is basedon stabilization of complex components like Neo1p andDop1p. Similar dependencies of complex subunits are notuncommon. For example, mutations or deletions in oneof the subunits of the fatty acid synthase (Fas1p/Fas2p)lead to the degradation of the non-mutated remainingsubunit (28), steady-state levels of clathrin heavy chain arereduced in clathrin light chain deletion mutants (�clc1) (29)and cdc50 mutant cells exhibit reduced amounts ofDrs2p (22,30). In the case of Neo1p loss in dop1-3 cells,our pulse-chase experiments show that Neo1p is morerapidly degraded when compared with isogenic DOP1cells (Figure S1). This degradation is not dependent onthe vacuolar protease Pep4p (Barbosa and Singer-Kruger,unpublished results). Rather, the turn-over appears to bemediated by the proteasome, as addition of the proteaso-mal inhibitor MG-132 largely delayed Neo1p degradation(Barbosa and Singer-Kruger, unpublished results). In addi-tion, in dop1-3 cells, Neo1p expression may be slightlydecreased, likely because of a rapid translational responsefrequently observed when mutant cells undergo stress.

Dop1p functions within the endomembrane system

In agreement with the subcellular localization of Neo1pand Ysl2p, which both have been previously localized toendosomes and the TGN (14–17), here we show thatDop1p colocalizes to a large extent with structures specif-ically labeled by bona fide endosomal markers introducedto the cell by endocytosis. Moreover, Dop1p colocalizedwith Hse1p and in vps27 cells the multiple GFP-Dop1pstructures collapsed into large clusters reminiscent of theendosomal class E compartment. In agreement with pre-vious studies (17), we also observed partial colocalizationof Dop1p with the late Golgi marker Sec7p. Consistentwith Dop1p’s localization to the TEN/TGN are severalphenotypes described for dop1 mutant alleles. First, in

the dop1-2 mutant the v-SNARE Snc1p and a modifiedsyntaxin, Sso1p, two markers that cycle between plasmamembrane, endosomes and the TGN, accumulated ininternal structures and were depleted from the plasmamembrane (17), suggesting that Dop1p is required forrecycling from endosomes. Second, conditional dop1mutants displayed vacuolar fragmentation (17) and dop1-3cells exhibited increased extracellular levels of the vac-uolar protease carboxypeptidase Y (CPY) (Barbosa andSinger-Kruger; Figure S4). Both phenotypes are highlycharacteristic for mutants with impaired trafficking withinthe TEN/TGN system as previously shown for ysl2 andneo1 mutants (14,15). Third, dop1-3 cells accumulatedelongated membrane processes and ring-like structuresas well as vacuoles, filled with membrane whorls, rem-iniscent of giant multivesicular bodies. Similar struc-tures have been previously identified in neo1 and ysl2mutants (14,15, this study) and interpreted as being theresult of impaired fission because of non-coordinatedactivity of Ysl2p, Arl1p and Neo1p (19). The related mor-phological alterations displayed by the dop1-3 mutantcorroborate Dop1p’s functional link to the Ysl2p-Arl1p-Neo1p network. The proposed role of yeast Dop1p inendosome biogenesis and trafficking is in excellent agree-ment with the postulated role of the Aspergillus nidulansorthologue DopA in cellular morphogenesis and filamen-tous growth, a process that is severely defective in thedopA mutant (31). Indeed, in filamentous fungi endocyticrecycling plays a key role in hyphal tip growth. The impli-cated early endosome compartment is highly dynamicand moves bidirectionally via cytoskeletal and motor pro-teins (32). Likewise, regulated endosomal cycling from theTEN has been proposed to control the plasma membranearea during cell division of higher eukaryotic cells and toensure that the plasma membrane regains its appropriatelipid and protein composition (33,34). The obvious con-nection between endosome dynamics, polarized growthand cytokinesis may also better integrate two other appar-ently discrete defects displayed by dopA/dop1 mutants,namely impaired nuclear movement in dopA mutants (31)and defective cellular organization of peripheral ER indop1-2 cells (17). It is tempting to speculate that Dop1porthologues are a key component of a conserved molec-ular machinery implicated in the directed movement ofseveral organelles during cell growth and cell division. Inconclusion, while the physical and functional connectionsof Dop1p to the TEN/TGN system are presently most obvi-ous, given the complex nature of the associated traffickingroutes, future studies will help to delineate in further detailDop1p’s exact contribution.

The Dop1p protein and its domain organization

The presence of several putative domains within Dop1porthologues led us to analyze their features in furtherdetail. Not surprisingly, none of the three regions analyzedwere able to restore viability to �dop1 cells. Still, theydisplayed specific protein interactions and localized todistinct subcellular compartments. Among the threedomains, the internal one appears to be highly critical

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for association with endomembranes. Interestingly, whenthe N- and C-terminal regions are missing, endosomesseem to artificially cluster, suggesting that the terminimight provide some sort of spacer or regulatory function.As the internal Dop1p fragment also bound well toNeo1p and Ysl2p, this region appears to be relevantfor these interactions. Yet, the loss of Ysl2p caused adiminished but still punctate GFP-Dop1p staining pattern.This could be because of mislocalization to the cytosol andsubsequent degradation or simply because of the reducedGFP-Dop1p levels (Figure 5B). As Dop1p interacts withNeo1p independently of Ysl2p, Neo1p’s role during Dop1precruitment might be more critical than that of Ysl2p.

Expression of the GFP fusion containing the N-terminusof Dop1p, a region with the highest sequence identityamong orthologues, resulted in a major cytoplasmicstaining with some residual dot-like structures. Thecoimmunoprecipitation studies further indicated that whilethe N-terminus can interact with Neo1p, it cannotrobustly interact with Ysl2p. Therefore, even if the Neo1pinteraction can take place, the N-terminus appears tocontribute only weakly to Dop1p membrane association.Although an impaired folding of the separate N-terminuscannot be excluded, the fact that the Neo1p interactionand a weak membrane association were still preservedsuggests that this domain is at least partially functional. Insummary, weak membrane interaction, likely via Neo1p,is the first function we could identify for the N-terminaldomain. The fact that the described dop1 alleles carrymutations in this region (this study, 17) strongly indicatesthat this portion is highly critical for Dop1p function.

Another region conserved in Dop1p orthologues withseveral adjacent leucine zipper-like repeats is locatedin the C-terminus (31). In the current study, we showthat this region mediates the formation of Dop1p dimersor oligomers. In vitro, using purified GST- and His-tagged fusions, the interaction of the C-termini wasshown to be direct. In vivo, among the three Dop1pdomains the C-terminus coprecipitated best with full-length Dop1p. Therefore, Dop1p homodimers can be gen-erated primarily via interactions between the C-terminaldomains. The N-terminal and internal domains may con-tribute to intermolecular binding, as two full-length Dop1pproteins coprecipitated more efficiently than Dop1p andthe C-terminus. However, as the N-terminal and internaldomains interact efficiently with Neo1p and Neo1p/Ysl2p,respectively, the former may also bind to full-length Dop1pvia Ysl2p and/or Neo1p. In any event, the large Dop1pcan form multimers, primarily through direct interactionsof leucine zipper-like domains in the C-terminus that mayadopt a coiled-coil structure. Intriguingly, microscopic anal-ysis suggested that the C-terminal fragment is localizedto the nucleus. Whether this localization is physiologicallyrelevant for Dop1p is presently unclear. The C-terminalconstruct might accumulate there because of lack or inac-cessibility of putative export signals, or because its rateof entry might exceed its rate of export. Similar to the

behavior of proteins with dual localization like transcrip-tion factors, it is possible that Dop1p (or a C-terminalfragment generated in vivo) undergoes nucleocytoplas-mic shuttling. A relocation from the TEN/TGN into thenucleus may occur under special physiological conditions.In fact, a potential role as transcriptional coactivator hasbeen originally postulated for A. nidulans DopA. In addi-tion to the conserved leucine zipper-like domains, DopAcontains an activation domain at the extreme C-terminuscharacteristic of CAAT/enhancer binding protein (C/EBP)family members. Although this sequence element is notobvious in other DopA orthologues including Dop1p (31),it may be hidden because of low sequence identity. Thephenotypes displayed by the dopA mutant suggested thatDopA is required for temporal upregulation of essentialdevelopmental regulators during cellular morphogenesis,highlighting a possible role in developmental program-ming and developmental gene expression (31). Therefore,nucleocytoplasmic shuttling might contribute to a regu-latory circuit that coordinates cell growth with endocyticrecycling to adjust membrane transport to the growthrequirements of the cell. Significantly, the single pointmutation in dopA and several amino acid changes inDop1-3p (Figure 4A) alter the leucine zipper-like dimeriza-tion domain. This is a strong evidence that it plays a keyrole in the function of Dop1p orthologues. Whether this isbecause of the ability to increase the avidity for its specificbinding partners (see below) and/or because of a potentialrole in the nucleus awaits further investigation.

Oligomeric Dop1p is linked to microdomains where

membrane remodeling occurs

The ability to form dimers or multimers is very commonamong proteins with a role in membrane bending anddeformation, with the membrane-shaping proteins of theBAR domain superfamily and clathrin representing promi-nent examples. Interestingly, in addition to Dop1p, at leastYsl2p is known to interact with itself and to exist in highmolecular mass complexes (16, Singer-Kruger, unpub-lished results). Neo1p and Arl1p may form oligomers, too,like other P-type ATPases and Arf family members such asPma1p and Arf1p (35,36). Thus, Dop1p, Ysl2p and Arl1pmay form a highly concentrated, tight meshwork com-posed of elongated rod-like molecules over the membranesurface where Neo1p is localized. Together with the asso-ciated coat components like clathrin and adaptors (19), thismicrodomain might ensure that membrane deformationsare selectively formed and stabilized at the places whereNeo1p induces membrane curvature by generating lipidasymmetry. The multivalent interactions within the matrixmight be required to provide specificity to the region ofthe organelle where bending occurs, to segregate appro-priate cargo into such budding intermediates and to linkthe forming structure to the correct cytoskeletal elements.Perhaps, this explains the high degree of redundancy thatseems evident for many transport steps, including thoseinvolving P4-ATPases, and may also account for thosecases where loss or mutation of one protein can be res-cued by overexpression of a homologue, an interaction

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partner, or a downstream component (see above). Ourstudies also suggest that Neo1p acts independently ofCdc50p proteins, as indicated by the large differences ingrowth phenotypes associated with the respective dele-tion mutants. Obviously, we have much to learn aboutthe exact roles of Dop1p, Neo1p and Ysl2p in membraneremodeling at the TEN/TGN interface and the temporalmechanisms of assembly during membrane bending.

Materials and Methods

StrainsYeast strains are listed in Table 1. Typically, they were derivatives ofRH1201 and were constructed by polymerase chain reaction (PCR)-basedintegration cassettes and homologous recombination and by crosses aspreviously described (19). Strains were grown at 25◦C to early logarithmicphase in standard media.

PlasmidsThe dop1-3 allele was generated in R. Kahn’s laboratory as follows: DOP1,subcloned into a LEU2-based plasmid, was mutagenized in vitro withhydroxylamine and transformed into �dop1 cells carrying a URA3-basedDOP1 plasmid. Transformants were replica plated onto SD-Leu plateswith 5-fluoroorotic acid and screened for growth at 25◦C and 37◦C. Theplasmid containing the dop1-3 allele was rescued from a colony thatgrew at 25◦C, but not at 37◦C, and retransformed to confirm the plasmiddependence of temperature-sensitive growth. The mutations found indop1-3 result in the following amino acid exchanges: L125S, S224L,E1438D, M1494I, Y1515H, C1580Y and E1638K. The strain BS1361 wasobtained by backcrossing YBB073 three times to a wild-type strain of MATalpha (corresponding to BS64). DOP1 was cloned by complementation ofthe temperature sensitivity of dop1-3 cells using a Ycp50-based genomiclibrary. The plasmid pSB131 contains an approximately 6.5-kb insert withthe DOP1 open reading frame and at least 500 bp of 3′ and 5′ non-translatedsequences. To subclone DOP1, two fragments (bp −347 to 577 and 3358 to5178, respectively) were amplified from pSB131 and inserted into pRS426using NotI/BamHI and BamHI/SalI, respectively, yielding pSB163. Theinternal fragment (bp 354-3549) was isolated from pSB131 and subcloned

Table 1: Strains used

Yeast strain Genotype Source

RH1201 MATa/α his4/his4 ura3/ura3 leu2/leu2 lys2/lys2 bar1-1/bar1-1 H. Riezman, GenevaBS25 MATa his4 ura3 leu2 lys2 ypt51::LYS2 bar1-1 (25)BS64 MATa his4 ura3 leu2 lys2 bar1-1 (25)BS694 MATa his4 ura3 leu2 lys2 ysl2::kanr bar1-1 (15)BS747 MATa/α his4/his4 ura3/ura3 leu2/leu2 lys2/lys2 ysl2::kanr /ysl2::kanr bar1-1/bar1-1 (15)BS862 MATa his4 ura3 leu2 lys2 neo1::kanr bar1-1 + pRS315-HA-NEO1 (14)BS915 MATa his4 ura3 leu2 lys2 neo1::kanr bar1-1 + pRS315-neo1-37 (14)BS917 MATa his4 ura3 leu2 lys2 neo1::kanr bar1-1 + pRS315-neo1-69 (14)BS957 MATa his4 ura3 leu2 lys2 vps27 bar1-1 This studyBS1021 BS747 + pSF316 (encodes ysl2-316) (15)BS1121 MATα ura3 leu2 lys2 YSL2::3-HA-HIS5 (S. pombe) bar1-1 (14)BS1361 MATα his4 ura3 leu2 lys2 dop1::kanr bar1-1 + pRS315-dop1-3 This studyBS1488 MATa his4 ura3 leu2 lys2 bar1-1 3-HA::NEO1 This studyBS1806 MATa/α his4/his4 ura3/ura3 leu2/leu2 lys2/lys2 dop1::kanr /dop1::kanr

bar1-1/bar1-1 + pRS315-dop1-3 This studySB71 MATa his4 ura3 leu2 lys2 cdc50::URA3 bar1-1 3-HA::NEO1 This studySB90 MATa his4 ura3 leu2 lys2 bar1-1 CDC50::13myc-kanr 3-HA::NEO1 This studySB121 MATa his4 ura3 leu2 lys2 bar1-1 CDC50::13myc-kanr This studySB142 MATα his4 ura3 leu2 lys2 dop1::kanr bar1-1 3-HA::NEO1 + pRS315-dop1-3 This studySB170 MATa his4 ura3 leu2 lys2 bar1-1 DOP1::3-HA-kanr This studySB201 MATa his4 ura3 leu2 lys2 neo1::kanr bar1-1 + pRS316-HA-NEO1 This studySB205 MATa his4 ura3 leu2 lys2 neo1::kanr bar1-1

DOP1::TAP-URA3 (K. lactis) + pRS315-HA-NEO1 This studySB284 MATa his4 ura3 leu2 lys2 neo1::kanr bar1-1 GFP::DOP1 + pRS316-NEO1 This studySB286 MATa his4 ura3 leu2 lys2 neo1::kanr bar1-1 GFP::DOP1 + pRS315-neo1-37 This studySB289 MATa his4 ura3 leu2 lys2 neo1::kanr bar1-1 GFP::DOP1 + pRS315-neo1-69 This studySB296 MATa his4 ura3 leu2 lys2 neo1::kanr ysl2::natr bar1-1

DOP1::TAP-URA3 (K. lactis) + pRS315-HA-NEO1 This studySB306 BS694 + pRS315-HA-NEO1 This studySB323 MATα his4 ura3 leu2 lys2 dop1::kanr bar1-1 3-HA::NEO1 + pRS315-DOP1 This studySB330 MATα his4 ura3 leu2 lys2 bar1-1 dop1::kanr + pRS315-DOP1 This studySB340 MATa/α his4/his4 ura3/ura3 leu2/leu2 lys2/lys2

ysl2::kanr /ysl2::kanr bar1-1/bar1-1 3-HA::NEO1/3-HA::NEO1 This studySB375 MATα his4 ura3 leu2 lys2 bar1-1 dop1::kanr + pRS315-PADH1-GFP-DOP1 This studySB434 MATa/α his4/his4 ura3/ura3 leu2/leu2 lys2/lys2

ysl2::kanr /ysl2::kanr bar1-1/bar1-1 GFP::DOP1/GFP::DOP1 This studySB463 MATa his4 ura3 leu2 lys2 neo1::kanr bar1-1 + pRS316-HA-Neo1-69p This studySB465 MATa his4 ura3 leu2 lys2 neo1::kanr bar1-1 + pRS316-HA-Neo1-37p This studyySL089 MATa his3�1 ura3�0 leu2�0 met15�0 YDR170c::YDR170c-mCh-kanr (37)ySL097 MATa his3�1 ura3�0 leu2�0 met15�0 YHL002w:: YHL002w-mCh-kanr (37)yBB073 MATa his3 leu2 lys2 ura3 met15 ydr141c::kanr pCEN-LEU2-ydr141cts R. Kahn, Atlanta

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into pSB163 with BamHI/SpeI, yielding pRS426-DOP1. The plasmidspRS315-DOP1 and pRS316-DOP1 were derived from pRS426-DOP1 usingNotI/SalI. To generate pRS315-PADH1-GFP-DOP1, two PCR fragments(bp 3-577 and 3358-5480) were inserted into pRS315 with NotI/BamHIand BamHI/SalI, respectively. The internal DOP1 fragment was insertedas described. Finally, a PCR fragment encoding the ADH1 promoter andGFP was amplified from pYM-N9 (Euroscarf collection) and subcloned viaSacI/NotI. The plasmid pRS315-Cterm-Dop1 is based on pRS315-PADH1-GFP-DOP1, but contains a NotI/SalI fragment (bp 4397-5480) encoding theC-terminus. The plasmids pRS315-Nterm-Dop1 and pRS315-internal-Dop1contain a NotI/SalI fragment with the DOP1 terminator (bp 5098-5480).Fragments encoding the Dop1p N-terminus (aa 2-502) or the centralpart (aa 502-1466) were introduced via NotI/NheI. The plasmid pGEX-4T-3-CtermDop11443−1698 contains a fragment encoding residues 1443-1698 ofDop1p, cloned via BamHI/NotI; pET15b-CtermDop11443−1698 contains thesame fragment, subcloned via XhoI/BamHI. Sequences of oligonucleotidesare available upon request. All constructs with PCR fragments wereconfirmed by sequencing. The plasmids pRS426-NEO1, pRS426-DRS2,pBS313, pRS426-ARL1, pRS315-HA-NEO1 and pGEX5-Ypt7 have beendescribed (14,15).

ImmunoprecipitationsImmunoprecipitations of Cdc50p-Myc and HA-Neo1p were performed asdescribed (21) with minor modifications. Cell extracts were preclearedby centrifugation at 400 × g for 5 min and centrifuged at 100 000 × g

for 1 h. Pellets were homogenized by dounce homogenization. Insol-uble material was removed by recentrifugation at 100 000 × g. Forimmunoprecipitations, the supernatants were incubated with either rabbitα-Myc or rat α-HA antibodies and protein A–Sepharose. Other coim-munoprecipitations were performed as previously described (14). Whenindicated, lysates were centrifuged at 100 000 × g for 1 h to removenon-solubilized proteins. Immunoprecipitated proteins were detected byimmunoblotting.

Shift assaysCells were grown at 25◦C to early logarithmic phase and shifted to 37◦C forthe indicated times. At each time-point, 1.7 optical density (OD)600 units ofcell were harvested, lysed with glass beads and processed for SDS–PAGEand immunoblotting. To quantify GFP-Dop1p in cell lysates, it was enrichedby immunoprecipitations and detected by immunoblotting using an IRDye®800-conjugated secondary antibody. For quantitations, membranes werescanned at 800 nm using the Odyssey Infrared Imaging System; signalswere quantified using Odyssey software (LI-COR Biosciences).

AntibodiesFor immunoprecipitations, rabbit α-Myc (A-14, Santa Cruz Biotechnologies),rat α-HA (3F10, Roche) and rabbit α-GFP (Invitrogen) were used. Forimmunoblotting, rabbit α-TAP (Open Biosystems), mouse α-HA (16B12,Covance), mouse α-Myc (9E10, Calbiochem), rabbit α-GFP (Invitrogen),mouse α-PGK (Molecular Probes), mouse α-Pma1p (40B7, Abcam),rabbit α-Ysl2p (15), rabbit α-Drs2p (T. Graham, Nashville) and mouseα-Chc1p (S. Lemmon, Miami) were used as primary antibodies. Secondaryantibodies were conjugated to alkaline phosphatase (KPL) or IRDye® 800(Rockland).

Pull-down experiments with glutathione

S-transferase fusionsSoluble GST and polyhistidine (His) fusion proteins were purified accordingto the manufacturer’s instructions. GST pull downs were performed aspreviously described (19).

MicroscopyIndirect immunofluorescence was performed as described (14). GFP-tagged Dop1p was observed in cells resuspended in 100 mM KPi,

pH 6.5, 1.2 M sorbitol, mixed with 1.6% low melting agarose. Nucleiwere stained with Hoechst-33342 (3 μg/mL). Endosomes were labeledby incubating cells at 0◦C for 1 h with Alexa594-α-factor [1.4 μg/mL,synthesized according to Ref. (24)] or FM4–64 (0.025 mM, Invitrogen).Subsequently, cells were washed with SD medium, shifted to 15◦C for30 min (wild-type cells) or 30◦C for 40 min (�ypt51 cells) and viewed byfluorescence microscopy.

Transmission electron microscopyYeast cells were cryoimmobilized by high-pressure freezing and preparedfor ultrastructural analysis as described (14).

Acknowledgments

We are grateful to B. Bowzard and R. Kahn for providing the dop1-3 allele,T. Graham, S. Lemmon and S. Leon for antisera and strains and D. Rais forthe recombinant GST-Cterm-Dop1 fusion. We acknowledge H. Rudolphfor critical reading of the manuscript and helpful discussions. This workwas supported by the Marie Curie Research Training Network (grant 5330,FP6-2002-Mobility-1) and the Deutsche Forschungsgemeinschaft (SI 635).

Supporting Information

Additional Supporting Information may be found in the online version ofthis article:

Figure S1: HA-Neo1p is destabilized in dop1-3 cells upon shift to 37◦C.

Isogenic DOP1 and dop1-3 cells expressing HA-Neo1p from a CEN-basedvector were labeled at 25◦C for 30 min with [35S]-methionine, and chasedfor 30, 60, 120 and 180 min at 37◦C. At each time-point, HA-Neo1pwas immunoprecipitated under denaturating conditions using a rat α-HAantibody. Samples were analyzed by SDS–PAGE and autoradiography.HA-Neo1p signals were quantified with the phosphorimager. The t1/2 ofHA-Neo1p was determined from five independent experiments.

Figure S2: The lethality of Δdop1 cells can neither be rescued

by NEO1 overexpression, nor complemented by overexpression of

Dop1p fragments. A) he lethality of �dop1 cells cannot be rescued byNEO1 overexpression. �dop1, cells transformed with pRS316-DOP1 andLEU2-based vectors encoding Neo1p, GFP-Dop1p or Dop1p as indicatedwere incubated at 25◦C as serial dilutions on minimal SD minus leucineplates with or without 5-fluoroorotic acid (5-FOA) to counterselect forthe pRS316-DOP1 plasmid. B) �dop1 cells transformed with pRS316-DOP1 and pRS315-based vectors expressing full-length GFP-Dop1p or theindicated GFP-Dop1 fragments were incubated at 25◦C as described in (A).

Figure S3: GFP-Dop1p colocalizes with the endosomal marker Hse1p

in wild-type cells and the class E compartment in vps27 cells, and

it partially colocalizes with the TGN marker Sec7p. GFP-Dop1p wasexpressed from a CEN-based vector in cells expressing Hse1p-mCherryfrom the chromosome (ySL097) (A), in vps27 cells (BS957) (B) and incells expressing Sec7p-mCherry from the chromosome (ySL089) (C).Observation of cells was by fluorescence (A–C) and Nomarski (B) optics.*, overlapping structures. Bar, 5 μm.

Figure S4: Mutant dop1-3 cells exhibit increased levels of CPY in the

extracellular space. Serial dilutions of isogenic NEO1, neo1-69, DOP1and dop1-3 cells were spotted onto yeast peptone dextrose (YPD) platesand grown for approximately 30 h at 25◦C with a nitrocellulose membraneplaced on top. Subsequently, cells were washed off the filter. ExtracellularCPY was detected by immunoblotting using a mouse α-CPY antibody(Invitrogen). Mutant neo1-69 cells served as an internal control, as theywere previously shown to exhibit defects in CPY sorting (14).

Please note: Wiley-Blackwell are not responsible for the content orfunctionality of any supporting materials supplied by the authors.Any queries (other than missing material) should be directed to thecorresponding author for the article.

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