Golvesin-GFP fusions as distinct markers for Golgi and post-Golgi vesicles in Dictyostelium cells

Original article

Golvesin-GFP fusions as distinct markers for Golgi and post-Golgi vesicles in Dictyostelium cells

Natalie Schneidera, Jean-Marc Schwartza, Jana Köhlera, Michael Beckera, Heinz Schwarzb,Günther Gerischa*a Max-Planck-Institut für Biochemie, D-82152 Martinsried, Germany

b Max-Planck-Institut für Entwicklungsbiologie, D-72076 Tübingen, Germany

Received 7 July 2000; accepted 25 October 2000

Golvesin is a new protein associated with membranes of the Golgi apparatus and post-Golgi vesicles inDictyostelium cells. An internal hydrophobic sequence of 24 amino-acid residues is responsible for anchoringgolvesin to the membranes of these organelles. In an attempt to visualize organelle dynamics in vivo, wehave used specific antibody and other labels to localize golvesin–green fluorescent protein (GFP) constructsto different cellular compartments. With a GFP tag at its N-terminus, golvesin shows the same localizationas the untagged protein. It is transferred to two post-Golgi compartments, the endosomal and contractilevacuole systems. Endosomes are decorated with GFP–golvesin within less than 10 min of their internalisa-tion, and keep the label during the acidic phase of the pathway.Blockage of the C-terminus with GFP causes entrapment of the protein in the Golgi apparatus, indicating thata free C-terminus is required for transfer of golvesin to any of the post-Golgi compartments. TheC-terminally tagged golvesin proved to be a reliable Golgi marker in Dictyostelium cells revealing protrusionof Golgi tubules at peak velocities of 3 to 4 µm·s–1. The fusion protein is retained in Golgi vesicles duringmitosis, visualizing Golgi disassembly and reorganization in line with cytokinesis. © 2000 Éditionsscientifiques et médicales Elsevier SAS

contractile vacuoles / Dictyostelium / endosomes / green fluorescent protein / Golgi dynamics

1. INTRODUCTION

Fluorescent markers applicable in vivo have dis-closed an unexpected repertoire of dynamic changes inthe Golgi apparatus (GA) of mammalian cells. Mostobvious are tubules that are not only found in brefeldinA-treated cells (Klausner et al., 1992), but also detect-able in untreated cells by the use of NBD–ceramide(Cooper et al., 1990), or green fluorescent protein

(GFP)-tagged Golgi markers (Sciaky et al., 1997). TheGolgi tubules are considered to mediate anterogradetransport from the GA to the plasma membrane or intolysosomes, and also retrograde transport to the endo-plasmic reticulum (ER) (Farquhar and Hauri, 1997;Hirschberg et al., 1998; Lippincott-Schwartz et al.,1998). The Golgi elements move along microtubules(Ho et al., 1989; Kreis et al., 1997; Lippincott-Schwartz,1998; Toomre et al., 1999). This movement can bedriven by at least five different kinesins (Robertson andAllan, 2000), or by cytoplasmic dynein (Corthésy-Theulaz et al., 1992; Burkhardt et al., 1997). In mousestem cells, elimination of cytoplasmic dynein heavy

* Correspondence and reprints: fax: +49 89–8578–3885.E-mail address: [email protected] (G. Gerisch).

Biology of the Cell 92 (2000) 495−511© 2000 Éditions scientifiques et médicales Elsevier SAS. All rights reservedS0248490000011023/FLA

Golgi and post-Golgi dynamics Schneider et al.

chain causes the GA to vesiculate, apparently drivenby unbalanced, plus-end directed kinesin activity(Harada et al., 1998).

In Dictyostelium, as in mammalian cells, the GA isknown to be connected to the centrosome (Wuestehubeet al., 1989; Zhu et al., 1993), but its organization wasessentially unknown. In particular, there has been ademand for a reliable marker to study Golgi dynamicsin vivo. Here we use GFP fusions of a new protein,golvesin, to study Golgi and post-Golgi dynamics ininterphase and mitotic cells of Dictyostelium discoideum.The golvesin gene has fortuitously been cloned in asearch for proteins that regulate cytoskeletal functionsin Dictyostelium. Although there was no evidence forassociation with the cytoskeleton, the protein appearedto be of interest because its intracellular distributiondepended on the position of a GFP tag. With GFP atthe C-terminus, golvesin proved to accumulate in theGA. Using this construct we show that the GA inDictyostelium undergoes rapid shape changes, includ-ing phases of extensive tubulation.

Placing the GFP tag to the N-terminus enabledgolvesin to localize, in addition to the GA, to twopost-Golgi compartments: vesicles of the endosomalpathway, and contractile vacuoles. The contractilevacuole network is an osmoregulatory organelle dis-tinct from the endosomal system (Heuser et al., 1993;Gabriel et al., 1999). Endocytosis is essential for nutri-tion, since Dictyostelium cells grow by digesting phago-cytized bacteria. Laboratory strain cells incorporatealso liquid nutrients by macropinocytosis (Hacker etal., 1997). Accordingly, Dictyostelium cells are suppliedwith an efficient system for actin-based nutrient up-take, endosome processing, and exocytosis (reviewedby Maniak, 1999). We show that the N-terminallytagged golvesin most strongly associates with themembranes of endosomes, and can be used to monitorvesicle processing in the endocytic pathway. Togetherour data on the two golvesin constructs provide evi-dence for the capability of an appropriately placed GFPtag to precisely block a specific step in protein translo-cation, here at the exit from the GA.

2. MATERIALS AND METHODS

2.1. Cloning and sequencing of golvesin

The 3’-region of the golvesin gene was obtained as asequence flanking the site of vector integration in amutant derived from the D. discoideum AX2-214 strainby restriction enzyme-mediated insertion (REMI;Kuspa and Loomis, 1992) as modified by Morio et al.(1995). The 5’-region was sequenced using an EcoRVclone of genomic DNA from D. discoideum strain AX2.The centre of the coding region was amplified by PCRfrom a λgt11 cDNA library of strain AX3 (courtesy

Richard Kessin, Columbia University, New York), us-ing a gene-specific and a λ primer. For sequence analy-sis, Wisconsin Package Version 9.0 of the University ofWisconsin Genetics Computer Group was used.

2.2. Vector construction

For the overexpression of untagged golvesin, thegenomic golvesin-encoding sequence of the D. discoi-deum AX2-214 strain was cloned into the EcoRI site ofthe pDEXRH vector (Faix et al., 1992). To expressGFP–(N)golvesin, the golvesin sequence was clonedinto the EcoRI site of the pDEX-GFPN vector (Westphalet al., 1997), with golvesin connected to S65T-GFP(Heim and Tsien, 1996) by the linker KLEFK.

Golvesin(C)–GFP was expressed using a pDEXRHvector with the S65T-GFP encoding sequence insertedinto the HindIII site in a way that the hexapeptideEFKKLK linked golvesin to the GFP-encoding se-quence (pDEX-GFPC; Westphal, unpublished). The ge-nomic golvesin sequence preceded by AAA andending in front of the stop codon was cloned into theEcoRI site of this vector.

2.3. Cell culture and transformation

D. discoideum strain AX2-214, here designated aswild type, or transformants of this strain were culti-vated in 10 mL of nutrient medium in polystyrene Petridishes. Expression vectors were introduced by elec-troporation into the genome of AX2 cells or of HG1668cells expressing GFP–α-tubulin (Neujahr et al., 1998).Transformants were selected for G418 resistance asdescribed by Neujahr et al. (1998). To cells producinggolvesin(C)–GFP and GFP–α-tubulin, 20 µg·mL–1 ofG418 and 10 µg·mL–1 of blasticidin S were added.

2.4. Fluorescence microscopy of living cells

Cells settled on a 5 × 5 cm glass coverslip within aplastic ring of 40 mm diameter were washed twicewith 17 mM K/Na-phosphate buffer, pH 6.0. For theobservation of mitosis, Klebsiella aerogenes was added asa nutrient. Immediately before microscopy the cellswere compressed by a 0.2 mm layer of 2% agarose inphosphate buffer (Yumura et al., 1984). For phagocyto-sis, D. discoideum cells were incubated with livingSaccharomyces cerevisiae strain TH2-1B, a gift of W.Tanner, Regensburg. All experiments were performedat 23 ± 2 °C.

Confocal fluorescence images of live cells were takensimultaneously with phase-contrast images using anLSM 410 microscope (Zeiss), and a 100× 1.3 FLUAR, or100× 1.3 Plan NEOFLUAR objective (Zeiss). For theexcitation of S65T-GFP or NBD-ceramide, the 488 nmband of an argon-ion laser was used together with a515–565 nm filter for emission. For simultaneous re

496 Biology of the Cell 92 (2000) 495–511


cording of GFP and neutral red, FM4-64, or TRITC–dextran, the 488 nm laser band was used together withthe 543 nm band of an He–Ne laser, and emission wassplit by the use of a 510–525 nm filter for GFP, and a570 nm high-pass filter for the fluorescence of otherdyes. Cells were incubated in nutrient medium con-taining 2 to 5 mg·mL–1 of TRITC–dextran (Sigma), andsubsequently compressed with an agar layer equili-brated with medium containing TRITC–dextran.Acidic vesicles were visualised by incubating the cellsin 0.5 µΜ neutral red (Sigma) in nutrient medium(figure 8), or 2.5 µM in 17 mM K/Na-phosphate buffer,pH 6.0 (figure 10). FM4-64 (Molecular Probes) was di-luted 1:2000 from a 1 mg·mL–1 stock solution in DMSO.

For staining with NBD–C6–ceramide (MolecularProbes), a complex of the ceramide with lipid-free BSA(Sigma) was prepared essentially as described by Pro-vance et al. (1993), and Ladinsky et al. (1994), but theBSA concentration was increased up to 34 mg·mL–1 in17 mM K/Na-phosphate buffer, pH 6.0. Cells were in-cubated for 1 h on ice in the dark with 5 µM of theBSA-bound ceramide, and were subsequently com-pressed with a glass coverslip for confocal imaging atroom temperature.

For labelling of contact sites A (csA) on the cellsurface, cells expressing golvesin(C)–GFP were starvedfor 6 h in 17 mM K/Na-phosphate buffer, pH 6.0,washed, and labelled in the buffer on ice for 1 h withthe monoclonal antibody (mAb) 41-71-21 specific forthe protein moiety of the csA glycoprotein, and afterwashing, for 30 min in Cy3-conjugated goat anti-mouse IgG (Jackson ImmunoResearch). Immediatelyafter washing, cells were scanned in the phosphatebuffer at 23 °C.

For velocity measurements (figure 5), an Axiovertmicroscope (Zeiss) equipped with a SIT C2400-08 cam-era (Hamamatsu), and software as described by Neu-jahr et al. (1998) was employed. Images were obtainedat intervals of 0.4 s, each one averaged over 4 frames ofthe SIT camera. The data were processed using AVS(Advanced Visual Systems, Waltham, MA), orCorelDraw 8.0 together with Adobe Photoshop soft-ware. Vesicle movement was interactively measuredusing a custom designed software.

2.5. Fluorescent labelling of fixed cellswith monoclonal antibodies or WGA

For the production of anti-golvesin mAb 275-392-5,fragments comprising 30 kDa or 48 kDa of theC-terminal golvesin region were bacterially expressedas GST-fusion proteins, and purified from inclusionbodies. Balb/c mice were immunized with mixtures ofthese fragments by alternating intraperitoneal injec-tions with aluminium hydroxide or Bordetella pertussisantigen as adjuvants.

�-Tubulin was labelled with polyclonal rabbit anti-bodies, a gift from G. Marriott (Martinsried), O–glyco-sylated proteins with mAb 24-210-2 or 40-62-5(Bertholdt et al., 1985), protein disulfide isomerasewith mAb 159-387-4 provided by C. Heizer, calnexinwith mAb 270-390-2, a gift from M. Ecke, coronin withmAb 176-3-6 (De Hostos et al., 1991), V-ATPase Asubunit with mAb 221-35-2 (Jenne et al., 1998), com-mon antigen 1 of lysosomal enzymes with mAb 221-450-6, and vacuolin with mAb 264-323-7, both giftsfrom M. Maniak.

For fluorescent labelling, cells attached to glass cov-erslips were washed in 17 mM K/Na-phosphate buffer,pH 6.0, and, either directly or after 15 min of agaroverlay, were fixed with picric acid/formaldehyde,followed by post-fixation in 70% ethanol according toHumbel and Biegelmann (1992). The fixed cells wereimmunolabelled using TRITC-conjugated goat anti-rabbit IgG, and TRITC- or Cy2-conjugated goat anti-mouse IgG (Jackson ImmunoResearch) as secondaryantibodies. For labelling with wheat germ agglutinin(WGA), the cells were incubated for 30 min with2 µg·mL–1 Texas red-conjugated WGA (MolecularProbes) in phosphate-buffered saline, pH 7.4.

For confocal images of WGA- or antibody-labelledcells, the 488 nm line of an argon-ion laser and a510–525 nm emission filter for GFP and Cy2, the543 nm line of a He–Ne laser and a 570 nm high-passemission filter for TRITC, or a 590–610 nm band passfilter for Texas red were used. Fluorescence of DAPI(Sigma) was recorded using the 364 nm line of anargon-ion laser and a 397 nm high-pass filter togetherwith a 560 nm low-pass dichroic mirror.

2.6. Electron microscopy

Cells were fixed in a freshly prepared ice-cold mix-ture of 2% glutaraldehyde and 1% osmium tetroxide in140 mM phosphate buffer pH 7.2 for 1 h essentially asdescribed by Franke et al. (1969). After washing inwater, cells were block-stained with an aqueous solu-tion of 1% uranyl acetate, dehydrated with ethanol,and embedded in Epon. Sections were contrasted withuranyl acetate and lead citrate, and examined in aPhilips CM10 transmission electron microscope at60 kV using a 30 µm objective aperture.

2.7. Membrane extractionand immunoblotting

Membranes were suspended in 50 mM HEPESbuffer, pH 8.0, containing 50 mM NaCl, 1 mM EDTA,1 mM EGTA, and protease inhibitors, and extracted bythe addition of the same volume of either 200 mMNa2CO3 to increase pH to 10.3, 2 M NaCl, or 2% TritonX–100, as described by Becker et al. (1999). Proteinsfrom equal aliquots of pellets and supernatants were

Biology of the Cell 92 (2000) 495–511 497


resolved by SDS–PAGE in 12% gels, blotted, and la-belled either with mAb 264-449-2 against GFP, a giftfrom M. Maniak, or with mAb 275-392-5 againstgolvesin. Goat anti-mouse IgGs conjugated with alka-line phosphatase (Jackson ImmunoResearch) wereused as secondary antibodies.

3. RESULTS

3.1. Sequence characteristics of golvesinand intracellular patterns of GFP fusionproteins

Golvesin is a 64 kDa protein that contains noN-terminal leader sequence but internal stretches ofhydrophobic amino acids, one of them consisting of 24residues (figure 1A). Two clusters of basic amino acidsimmediately precede this hydrophobic region. TheN-terminal region from residues 12 to 75 consists of analmost homopolymeric asparagine stretch. Asparagineor glutamine homopolymers of unknown function areoften found in Dictyostelium proteins (Kimmel andFirtel, 1979; Shaw et al., 1989). Disregarding this aspar-agine stretch, golvesin shows no apparent homology toother proteins in the database, except for a marginalidentity of 22% residues between positions 123 and 421to the outer membrane protein Omp6 of Helicobacterpylori (accession number AADO 07298).

To localize golvesin in living cells, the protein wastagged either at its N- or C-terminus with GFP. Thetwo fusion proteins, GFP–(N)golvesin and golvesin-(C)–GFP, showed different patterns of intracellular lo-calization (figure 1B,C). In the following chapter wedemonstrate that golvesin(C)–GFP accumulates in theGA, while GFP–(N)golvesin is targeted to endosomesand contractile vacuoles.

The hydrophobic sequence of 24 amino-acid resi-dues is long enough to span the membrane. In order toshow that this sequence acts as a membrane anchor, itwas deleted in N-terminally and C-terminally taggedgolvesin. In both cases deletion of this hydrophobicdomain resulted in a complete loss of binding to anyorganelle membrane (figure 1D,E). The deleted proteinwas distributed within the cytoplasm. With the GFPtag at the C-terminus, it accumulated in the nucleus,probably because the basic cluster of amino acids 84 to94 acted in this construct as a bipartite nuclear localiza-tion signal. The N-terminal GFP may mask this signalin the other construct.

Fractionation of cell lysates confirmed the conclu-sions drawn from the sequence and the fluorescenceimages obtained in vivo. The untagged golvesin wasrecovered in the 100 000 × g pellets (figure 1F). Upontreatment with 1% of Triton X–100, only a minorfraction of the protein was extracted. With salt, or athigh pH, extraction was negligible. N- or C-terminally

tagged golvesin behaved similarly (data not shown).Deletion of the hydrophobic domain resulted in asoluble protein that was partially degraded (figure 1G).These results indicate that golvesin is integrated intomembranes by its hydrophobic domain, and extractionby non-ionic detergent is prevented by charge interac-tions, most likely due to the basis stretch in front of thehydrophobic domain.

3.2. Golvesin(C)-GFP decoratesthe centrosome-associated Golgi complex

Position, shape and size of the GA in Dictyosteliumare illustrated in figure 2. The Golgi complex is typi-cally found in close apposition to the centrosome,which in turn is attached to the nucleus (figure 2A). TheGA is subdivided into several stacks, which are oftenbent in a U-turn with their concave surfaces facing thecentrosome (figure 2B). Each stack consists of up to sixsaccules whose lateral portions widen into ampoules,which are intricately connected to the corona ofvesicles surrounding the centrosome (figure 2C). Itshould be noted that the GA in Dictyostelium is difficultto fix; it was not appropriately preserved by methodsthat allow subsequent immunolabelling.

To visualize the GA by fluorescence microscopy, aGolgi marker established for mammalian cells,NBD–C6–ceramide (Lipsky and Pagano, 1985), wasintroduced into living cells. The organelle labelled withNBD–C6–ceramide in Dictyostelium corresponded tothe electron microscopic appearance of the Golgi stacksin size, subdivision into lobes, and location near to thenucleus (figure 3A). The ceramide persisted in the GAfor about 30 min, provided that its metabolism wasinhibited by depleting the cells of oxygen.

The organelle labelled by golvesin(C)–GFP in livingcells was indistinguishable in size and shape from theceramide-labelled one (figure 3B as compared to figure3A). To establish the specificity of golvesin(C)–GFPlocalization to the GA, we co-labelled cells with a set oforganelle-specific markers. Decoration of endosomeswith golvesin(C)–GFP was excluded by loading livingcells with TRITC–dextran. Fluorescent dextran is takenup by Dictyostelium cells through macropinocytosis(Hacker et al., 1997), and is released by exocytosis afteran average residence time of about 60 min (Cardelli etal., 1989; Aubry et al., 1993; Jenne et al., 1998). In orderto load vesicles at all stages of the endocytic pathway,cells were incubated with TRITC–dextran for 2 to 3 h.No coincidence of GFP- and TRITC-labelled vesicleswas observed (figure 3B).

Figure 3C localizes the golvesin(C)–GFP label close tothe microtubule organizing centre, this means adjacentto the centrosome as shown in the electron micro-graphs of figure 2. WGA is a marker of the GA inDictyostelium cells (Wuestehube et al. 1989; Zhu et al.,1993). This lectin binds to �-dimers of N–acetylglu



Figure 1. Sequence and localization of golvesin tagged at its N- or its C-terminus with GFP. (A) Sequence of golvesin based on thegenomic sequence, submitted to the DDBJ/EMBL/GenBank databases under accession number U89350. (B) Golvesin linked to GFP at itsN-terminus (GFP–(N)golvesin) localizes to numerous intracellular vesicles. (C) Golvesin tagged at its C-terminus (golvesin(C)–GFP) isconcentrated in a perinuclear organelle. The nuclei (N) appear as black areas in the confocal fluorescence images. (D) Elimination of the 24amino-acid residues constituting the long hydrophobic domain, with the tag at the N-terminus; (E) the same tagged at the C-terminus. (F,G)Extractability of golvesin and of a GFP-tagged deletion construct. Cell homogenates were fractionated into supernatant (S) and pellet (P) at100 000 × g in HEPES buffer, pH 8.0 (control), as described in Materials and Methods, either in the presence of high salt (1 M NaCl), incarbonate (pH 10.3), or with 1% detergent (Triton X–100). Blots were labelled with antibodies as indicated on bottom of the panels. (F)Full-length overexpressed golvesin; (G) GFP–(N)golvesin without the hydrophobic domain as in (D). Bars in the confocal images (B–E),10 µm.



cosamine, a constituent of O–linked oligosaccharides(Haynes et al., 1993; Mang, 1995); added to proteins inthe GA (Yoshida et al., 1984; Hohmann et al., 1987).Strong WGA label was found within the organelledecorated by golvesin(C)–GFP (figure 3D), and weakerlabel at dispersed vesicles and the plasma membrane.These post-Golgi locations of O––glycosylated proteinswere not detectably decorated with golvesin(C)–GFP.

Similar labelling patterns as with WGA were ob-tained with mAb 40-62-5, an antibody specific forO–linked oligosaccharides (Bertholdt et al., 1985),whose binding to wild-type glycoproteins is inhibitedby free N–acetylglucosamine (Bozzaro and Merkl,1985). Like WGA, this antibody bound to the organellemarked by golvesin(C)–GFP. In figure 3E the overlap-ping areas are shown in yellow, and areas of un-matched GFP fluorescence in green. These data areconsistent with the view that golvesin(C)–GFP deco-rates the entire GA, whereas WGA and antibody leaveout the cis-Golgi compartments.

MAb 24-210-2 recognizes O–glycosylated epitopesselectively on two developmentally regulated glyco-proteins, the contact site A cell-adhesion protein anddajumin, a constituent of the contractile vacuole com-plex (Gabriel et al., 1999). We have taken advantage ofthe expression of both proteins after about 5 h of

starvation in order to label them on their transitthrough the GA. Comparison of figure 3F and G showsthat the O–glycosylated proteins accumulate firstwithin the organelle decorated with golvesin(C)–GFP.

To examine whether golvesin(C)–GFP co-localizeswith the cisternae of the ER, we labelled cells withantibody against the ER resident protein disulfideisomerase (Monnat et al., 1997). A very faint GFPfluorescence overlapped with the antibody label of theER (figure 3H). In summary, golvesin(C)–GFP high-lights the GA in living Dictyostelium cells, and distin-guishes it from the ER, which is faintly labelled, andfrom the endosomes, which remain unlabelled.

3.3. Golgi dynamics in Dictyostelium cellsvisualized with golvesin(C)–GFP

The state of the GA varied in Dictyostelium cellsbetween a compact organization to a dispersed struc-ture, sometimes changing from one state to the otherwithin less than 1 min (figure 4A). In the dispersedstate, vesicles often represented the thickened ends ofthin tubules that connected them to the central portionof the Golgi complex. These tubules appeared or dis-appeared in confocal sections from one image to thenext (figure 4B), indicating that they were seen only

Figure 2. Organization of theGolgi complex in D. discoideum.C, centrosome; GS, Golgi sac-cules; M, mitochondria; N, por-tions of the nucleus. (A) Stacks orsingle saccules of the GA near tothe centrosome that is associatedwith a tipped extension of thenucleus. (B) Bent stacks surround-ing the centrosome, typical of theGolgi complex in Dictyostelium.(C) Vesiculate interspace betweena Golgi stack and the centrosomeat high power of magnification.Bars, 500 nm.



Figure 3. The Golgi complex of Dictyos-telium in fluorescence images and thelocalization of golvesin(C)–GFP. (A)NBD–C6–ceramide label in confocal im-ages (green) superimposed to phase-contrast images (blue), illustratinglocation of the ceramide-labelled com-plexes close to the nuclei. (B–H) double-labelled cells in phase-contrast andconfocal fluorescence images. In theright panels, golvesin(C)–GFP is pseudo-colored in green, other labels are in red.Regions of overlap appear in yellow. (B) Aliving cell with labelled endosomes 2 hafter pre-incubation with TRITC–dextran.The golvesin(C)–GFP label coincides withthe rosette shape of the GA in figure 2B.(C) Antibody label of �-tubulin showingthe golvesin(C)–GFP label adjacent to themicrotubule organizing centres in a bi-nucleate cell. (D) Label of Texas red-conjugated wheat-germ agglutinin, aGolgi marker in Dictyostelium; (E) label ofmAb 40-62-5 against multiple O–glycosy-lated proteins; (F,G) mAb 24-210-2against O–linked oligosaccharides on de-velopmentally regulated proteins after4.5 h (F), or 5.5 h (G) of developmentunder starvation conditions. (H) Antibodylabel of protein disulfide isomerase visu-alizing the endoplasmic reticulum. Cen-tres of the nuclei are indicated by redcircles when recognizable in the phase-contrast images. Bars, 10 µm.



when exactly in focus. The tubules were linear, curvi-linear, or bent in a hairpin. Some extended to the cellperiphery, others surrounded the nucleus (figure 4C).The Golgi tubules reached an average length of 6 µm;the length of the longest tubule measured in a flattenedcell was 13 µm.

Microtubules of Dictyostelium cells are in a continu-ous waving movement. They emanate from a cen-trosome and are bent at the periphery of the cells.Some of the microtubules align at their proximal por-tions with the surface of the nucleus to which thecentrosome is attached (figure 4D). In order to relateshape changes of the GA to microtubule organization,we analysed cells in which a strong label of golvesin-(C)–GFP was superimposed to a weak fluorescence ofGFP–α-tubulin. Analyses of time series indicated thatGolgi vesicles or tubules attached to, and sometimesalso bundled, the microtubules. In all interphase cellsexamined, at least a portion of the Golgi complexremained in close association with the centrosome,even in phases of extensive tubulation or vesiculation.Figure 4E gives an example of Golgi vesicles thatchanged their position in association with microtu-bules. Frames 30–40-s of figure 4E represent a period of10 s in which a compact Golgi structure is reconstitutedfrom dispersed vesicles by minus-end directed move-ment along microtubules, some of them associatedwith the nucleus.

For quantitative motility analysis, the positions ofvesicles were determined at intervals of 0.4 s. Phases ofslow movement with inconstant directionality alter-nated with phases of faster, straight movement (leftpanels in figure 5). The faster phases included shortvelocity peaks of 3 to 4 µm·s–1, with an average widthof 0.8 s at half maximum (right panels of figure 5). Most

Figure 4. Golgi dynamics in Dictyostelium cells visualized bygolvesin(C)–GFP. Cells expressing golvesin(C)–GFP (A–C), GFP–α-tubulin (D), or both of these GFP-fusion proteins (E) in confocalfluorescence images. The cell in (A) reveals phases of compactand dispersed Golgi organization in a continuous time-series withframe-to-frame intervals of 4 s (from left to right). In the stronglycompressed cell shown in (B) Golgi tubules are transiently seenthat connect vesicles to the central Golgi complex. The pair ofvesicles on top of the first frame turned out in the 40-s frame toform the end of a tubule. Other vesicles representing thickeningsof a Golgi tubule are recognized in the 82-s frame. (C) A binucle-ate cell showing a more compact and a more dispersed GA, thelatter on bottom of the image extends into a tubule closelyassociated with the nucleus. The cell in (D) is a reference labelledwith GFP–α-tubulin alone, showing only the bent microtubulesradiating from the centrosome. The cell in (E), double-labelled withgolvesin–(C)GFP and GFP–α-tubulin, reveals association of mov-ing Golgi vesicles with microtubules. Numbers in (B) and (D)indicate seconds. N, nuclei. The arrowhead at 463-s points toGolgi vesicles clustered around the end of a spindle rudiment.Bars, 10 µm (the bar in D also applies to C and E).



often these peaks of velocity were observed in direc-tions either straight from, or towards the central GA.This finding is consistent with the view that theseGolgi structures are transiently coupled to microtu-bules, being moved through plus- or minus-end di-rected motors.

3.4. Mitotic Golgi re-organization inDictyostelium

Golvesin(C)–GFP was retained at distinct vesiclesthrough all stages of mitosis (figure 6A). Re-associationof these vesicles began simultaneously with incision ofthe cleavage furrow, which coincided with disassemblyof the spindle (figure 6B). In previous work the use ofGFP–α-tubulin uncovered vigorous, saltatory cen-trosome movements throughout the post-mitotic phase(Neujahr et al., 1998). Microtubules follow in form ofcomet tails the centrosomes during their movement inchanging directions. Other microtubules are attachedto the nuclei as in interphase cells.

The role of centrosome movements in Golgi re-assembly was investigated by combining a faint labelof GFP–α-tubulin with the golvesin(C)–GFP label ofthe GA (figure 7). The tubulin label was sufficient tovisualize the spindle and centrosome positions, with-out masking the Golgi vesicles during their assembly.Frames 0–226-s of figure 7 document that up to post-telophase there was no significant attachment ofvesicles to the spindle, which became extensively bentas the centrosomes moved. Golgi re-assembly com-menced when the spindle already disintegrated. TheGolgi vesicles gradually increased in size by fusion,and the majority of them accumulated along the comettails behind the moving centrosomes, some also at thenuclear surface (frames 226–661-s). In frames 367-s and463-s of figure 7, remnants of the spindle are seen totraverse the nuclei, as reported previously (Neujahr etal., 1998). The free cytoplasmic ends of these remnantsbecame also decorated with Golgi vesicles, as indicatedby arrowhead in the frame 463-s of figure 7.

3.5. Localization of GFP–(N)golvesin toendosomes and contractile vacuoles

GFP–(N)golvesin is not only found at the Golgicomplex but also at other intracellular vesicle systemsas shown in figure 1B. Association of the N-terminallytagged golvesin with endosomes was monitored in livecells incubated with medium containing TRITC–dext-ran as a fluid-phase marker that is taken up by macro-pinocytosis. Within 2 min of uptake, endosomesbecame surrounded by clusters of vesicles, whichtransferred GFP–(N)golvesin to the endosome mem-branes. This is shown in figure 8A, where a freshlyincorporated macropinosome on top of the cell is freeof GFP–(N)golvesin, while previously formed ones aredecorated (frames 0–36-s). Vesicles carrying GFP–(N)golvesin in their membranes are seen in figure 8A toaggregate and to form a rosette around the new endo-some (frames 132-s and 150-s), while the fluorescentprotein is delivered to its membrane (frame 256-s).

Pulse-labelling with TRITC–dextran confirmed thedecoration of endosomes with GFP–(N)golvesin withina period of 8 min (figure 8B). Later on, this lumenalmarker became distributed to numerous vesicles, sug-gesting budding and fusion of the endosomes. Finallythe fluorescent golvesin became concentrated in dots atthe endosome surface, and some of the vesicles weredepleted of golvesin while they retained the fluores-cent dextran label in their lumen (arrowheads in figure8B). These golvesin-depleted vesicles appeared afterabout 1 h, at a time after which late endosomes releasetheir contents by exocytosis (Aubry et al., 1995), indi-cating that GFP–(N)golvesin is retrieved before thevesicles fuse with the plasma membrane.

Figure 5. Tracks and velocities of two Golgi vesicles (A,B).Numbered arrowheads in the left panels correspond to phases ofpeak velocities in the right panels. Zero points on the coordinatesdefine positions of the central GA. (A) A vesicle that departs fromthe central GA, showing phases of straight movement interruptedby tumbling. (B) A vesicle that finally returns to the GA, with similarpeak velocities in both directions. Positions of the vesicles (leftpanels) and their velocities (right panels) were recorded at inter-vals of 0.4 s. The errors of frame-to-frame speed measurementsdue to pixelation of the images are between 0.8 and 1.1 µm·s–1.



The stages at which vesicles of the endocytic path-way are decorated with GFP–(N)golvesin were speci-

fied using coronin and other stage-specific markers.Coronin, a protein of the WD40 family, assembles

Figure 6. GA and microtubules in mitosis. (A) Stages of cytokinesis in a cell producing golvesin(C)–GFP in phase contrast (top) andconfocal fluorescence images (middle). (B) Stages of cytokinesis corresponding to those in (A) in a cell producing GFP–α-tubulin. In orderto recognize details of Golgi and microtubule organization, the cells had to be compressed between glass and an agar layer. Under theseconditions mitotic cleavage is slower than in a fluid layer (Neujahr et al., 1997). Time in (A) is indicated in seconds. Bars, 10 µm.

Figure 7. Time series of a mitotic cell showing Golgi vesicle assembly in line with centrosome movements. The cell was labelled withgolvesin(C)–GFP, and weakly with GFP–α-tubulin, and shows the GFP fluorescence in green superimposed to phase-contrast images inblue. The cell was strongly compressed in order to force centrosomes to move in one plane. Numbers indicate time in seconds. Thearrowhead at 463-s marks Golgi vesicles at a stub of the spindle. Bars, 10 µm (magnification remained unchanged for the first fiveframes).



together with filamentous actin at phagocytic cups,and dissociates from the phagosomes within 1 min ofcomplete engulfment (Maniak et al., 1995). Phago-somes decorated with coronin remained free ofgolvesin (figure 8C to E). Only after the release ofcoronin, endosomes acquired a continuous coat ofGFP–(N)golvesin (figure 8F,G).

Acidic vesicles, recognized by the accumulation ofneutral red, were strongly decorated with GFP–(N)golvesin (figure 8H). Similarly, endosomes carryinga sulphated protein-linked carbohydrate epitope as acommon lysosomal marker (Freeze et al., 1984; Freezeet al., 1990) were labelled with this marker in theirlumen, and with GFP–(N)golvesin at their surface(figure 8I).

Specific markers of late endosomes are vacuolin Aand B, two peripheral membrane proteins that primethe endosomes for exocytosis (Rauchenberger et al.,1997; Jenne et al., 1998). On vesicles labelled withanti-vacuolin antibody, no golvesin was found (figure8J). Together our results indicate that golvesin associ-ates with endosomes only during an intermediatephase of their pathway, following the release of coro-nin and preceding the acquisition of vacuolins.

Membranes of the ER, forming a layer around thenucleus that is connected to cisternae in the peripheryof the cells, are brightly labelled with antibody againstcalnexin. These ER membranes contained GFP–(N)golvesin. However, other vesicles were morestrongly decorated, in accord with the prominent asso-ciation of GFP–(N)golvesin with endosomes (figure 8K).

The contractile vacuole network is a profusely devel-oped post-Golgi compartment in Dictyostelium cells(Heuser et al., 1993; Gabriel et al., 1999). Golvesin-decoration of the contractile vacuoles was assessedusing an antibody against the 70-kDa subunit of vacu-olar proton-ATPase (Jenne et al., 1998). This protonpump is more strongly associated with the contractilevacuole complex than with acidic endosomes (Fok etal., 1993; Temesvari et al., 1994). Figure 8L shows a largevacuole and smaller vesicles strongly labelled withantibody against the proton-ATPase and decoratedwith GFP–(N)golvesin. In living cells, FM4-64 insertsinto the plasma membrane and subsequently into themembranes of contractile vacuoles (Heuser et al.,1993). Labelling with this dye showed that the GFP–(N)golvesin was retained at the vacuolar membrane atall stages of the contraction cycle (figure 8M).

In order to compare golvesin decoration of endo-somes and contractile vacuoles, the endosomes wereloaded with TRITC–dextran, and the unlabelled con-tractile vacuoles were identified by their periodic activ-ity. The time series of figure 8N reveals that theendosomes are most strongly decorated with GFP–(N)golvesin.

3.6. Untagged golvesin distributes similar toGFP–(N)golvesin

The different localizations of N- and C-terminallytagged golvesin prompted us to determine the localiza-tion of untagged golvesin using monoclonal antibod-ies. However, the endogenous golvesin was notabundant enough to produce a distinct pattern ofimmunofluorescence labelling. Vesicles or patches dis-tributed through the entire cell were too faintly la-belled to identify the organelles (figure 9A). Therefore,we first assessed the suitability of the antibodies raisedby recognition of the two GFP-fusion proteins at theircorrect locations: golvesin(C)–GFP at the GA and theER, and GFP–(N)golvesin also at post-Golgi vesicles(figure 9B,C).

Subsequently, the untagged golvesin was unequivo-cally localized by overexpressing the full-length pro-tein under the same strong promoter, as for the GFP-fusion proteins. In accord with the localization of bothN-terminally and C-terminally tagged golvesin, part ofthe overexpressed protein overlapped with WGA labelof the GA, and some label was detected on the ER, inparticular on the layer surrounding the nucleus (figure9D). In addition, the overexpressed golvesin decoratedvesicles of various sizes, similar to the localization ofGFP–(N)golvesin to endosomes and contractile vacu-oles (figure 9E). These results indicate that a GFP tag atthe N-terminus does not prevent golvesin from beingtargeted to the post-Golgi organelles that it normallypopulates, whereas occupation of its C-terminus ar-rests golvesin in the GA.

3.7. Protein transport in cells expressinggolvesin(C)–GFP

The data shown in figure 9 imply that endogenousgolvesin resides in the GA only transiently, whereasstrongly overexpressed golvesin(C)–GFP is sequesteredin the Golgi complex. To examine whether occupationof the GA with the golvesin construct interferes withthe passage of other proteins, we established that bothtargeting of proteins to the plasma membrane and toendosomes proceed in cells that strongly expressgolvesin(C)–GFP (figure 10).

To assess passage from the GA to the cell surface,living cells were labelled with an antibody against thedevelopmentally regulated csA cell-adhesion protein.These cells were individually scanned and selected forbrilliant GFP fluorescence in the GA. The csA protein issynthesized after 5 h of starvation, and is O–glycosy-lated in the GA before being transported to the plasmamembrane (Hohmann et al., 1987). After labelling withthe antibody in the cold, the csA molecules clusteredinto patches and accumulated at sites of cell-to-cellcontact. In conclusion, the presence of golvesin(C)–GFP



Figure 8. Localization of GFP–(N)golvesin to endosomes and contractile vacuoles. In the confocal images, GFP fluorescence is shown ingreen, other labels in red, and areas of superimposed labels in yellow. Except for (A,M), phase-contrast images in deep blue aresuperimposed to the fluorescence images. (A) Time series of a cell incubated with TRITC–dextran to label macropinocytic vesicles. (B)Cells incubated for 3 min with TRITC–dextran. After the pulse-labelling of endosomes with this fluid-phase marker, cells were washed andincubated in phosphate buffer for the indicated times, and imaged under strong compression by agar overlay. The five cells shownexemplify the indicated periods after chase. TRITC–dextran loaded vesicles not decorated with golvesin are indicated in the 51 and 59-minframes by arrowheads. (C–G) GFP–(N)golvesin combined with antibody-labelling of coronin in five different cells fixed after the uptake ofyeast particles. Coronin decorates phagocytic cups and early phagosomes before they are decorated with GFP–(N)golvesin (C–E).Phagosomes surrounded by the golvesin construct are not decorated with coronin (F,G), while coronin typically associated with the leadingedge (F). (H) A living cell incubated with neutral red, a marker of acidic vesicles. The membranes of these vesicles are consistently labelledwith GFP–(N)golvesin. (I) Antibody-labelling of an epitope common to lysosomal glycoproteins in the lumen of golvesin-decorated vesicles(arrowhead). (J) Mutually exclusive labelling of vesicles with GFP–(N)golvesin, or antibody against vacuolin, a marker of late endosomes.(K) Labelling with antibody against calnexin. This ER-membrane marker overlaps with GFP–(N)golvesin (perinuclear layer and extensions ofthe ER appear in yellow). Numerous other vesicles strongly decorated with GFP–(N)golvesin are not labelled by the antibody (green). (L)Antibody-labelling of vacuolar proton-ATPase, a membrane protein strongly enriched at contractile vacuoles and weakly represented atendosomes. Superimposition of the antibody label to GFP–(N)golvesin distinguishes endosomes, with a weak antibody label in yellow, fromthe contractile vacuole system, with a dominating antibody label in red. (M) A living cell preincubated with FM4-64, a dye preferentiallylabelling the contractile vacuole. GFP–(N)golvesin coincides with the dye label at the membranes of two contracting vacuoles (0 time), ofthe same vacuoles after their contraction (30 s), and after their expansion and fusion (150 s). The cell had been preincubated with FM4-64for 30 s; the first frame was taken at 14 min after removal of the dye. (N) A cell pre-incubated for more than 2 h with TRITC–dextran tolabel endosomes at all stages of the pathway. The cell shows contraction of a vacuole (arrowheads) that was less strongly labelled byGFP–(N)golvesin than the dextran-filled endosomes. Antibodies used are specified in Material and Methods. Numbers in frames indicateseconds. Bars, 10 µm.



in the GA did not prevent the normal exposure of thecsA protein on the cell surface (figure 10A).

Vacuolar H+-ATPase is sorted into post-Golgivesicles of the contractile vacuole system and of the

endosomal pathway (Fok et al., 1993). Phagosomesneed to incorporate this ATPase into their membranesfor acidification of the contents, a requirement for thedigestion of engulfed bacteria or yeast particles. To

Figure 9. Labelling with antibody localizes untagged, overexpressed golvesin to Golgi and post-Golgi compartments. (A) A wild-type cell;(B) a cell overexpressing golvesin(C)–GFP; (C) a cell overexpressing GFP–(N)golvesin; (D,E) cells overexpressing untagged golvesin. Allcells were labelled with anti-golvesin antibody. In (A–C,E) the antibody label is coloured in red. Nuclei stained with DAPI in (A, D and E) arein blue. The GFP fluorescence in (B) and (C) is shown in green. In (D) the antibody label is in green and the WGA label in red. The wild-typecell in (A) shows indistinct antibody labelling due to weak expression of the endogenous golvesin. Golvesin(C)-GFP and GFP–(N)golvesin arecorrectly localized by the antibody to their respective positions, i.e. to the Golgi complex (B) and post-Golgi vesicles (C). Coincidence ofGFP and antibody labels appears in yellow. Superimposition of the golvesin-antibody and WGA labelled structures in (D) shows coincidenceof the two labels at the Golgi complex (yellow). The green contour of the nucleus coincides with the perinuclear ER layer labelled by theantibody. Areas of the plasma membrane labelled with WGA are not matched by the antibody label (red). The cell shown in (E) contains theoverexpressed, untagged golvesin in a multitude of intracellular vesicles. Bars, 10 µm.

Figure 10. Protein transport to the plasma membrane (A) and processing of endosomes (B–E) in the presence of a strong load ofgolvesin(C)–GFP at the GA. The fluorescence of golvesin(C)–GFP is shown in green, other fluorescence is in red, and phase-contrastimages of the cells in blue. (A) Developing cells show the csA protein on the cell surface, in particular at areas of cell-to-cell contact. Nofluorescence on the cell surface was observed in similarly labelled, undeveloped control cells (not shown). (B–D) Phagocytizing cellsloaded with living yeast and incubated with neutral red illustrate the acidification of phagosomes. In (B) neutral red is accumulated in formof a crescent between endosome membrane and yeast particle (arrowhead). In (C,D) a well-defined lumenal space is recognized aroundthe neck region between mother cell and bud of dividing yeast, as indicated by arrowheads. (E) After a period of 1 min the yeast cell shownin (D) became permeabilized as indicated by neutral red staining of its cytoplasm. Bars, 10 µm (the bar in (E) applies also to (B–D)).



examine whether the residence of golvesin(C)–GFP inthe GA is compatible with translocation of the vacuolarATPase to endosomes, the acidification of phagosomesloaded with living yeast was monitored by the use ofneutral red. The dye indicated acidification by itsaccumulation within the space surrounding the par-ticle surface (figure 10B–D). The killing of yeast in theacidic phagosomes was identified by cytoplasmicstaining of the particle (figure 10E). These results ob-tained in cells selected for their strong expression ofgolvesin(C)–GFP are consistent with the progression ofphagosomes to the acidic phase as observed in normalcells.

4. DISCUSSION

4.1. Golvesin, a membrane protein of Golgiand post-Golgi vesicles

Golvesin is found in the ER, the Golgi complex, andin two types of post-Golgi organelles: vesicles of theendosomal pathway, and the contractile vacuole com-plex. A fusion protein with GFP at the N-terminus ofthe golvesin sequence shows the same localization asthe untagged protein. However, a GFP tag at theC-terminus sequesters golvesin in the GA, suggesting afunction of the C-terminal region in the transfer topost-Golgi compartments.

An internal hydrophobic sequence of 24 amino-acidresidues is essential for the binding of golvesin toorganelle membranes (figure 1D,E, and G). This se-quence comprises primarily aliphatic residues and ischaracterized by two proline residues, one close toeach end (figure 1A). Two clusters of basic amino acidsare located on the N-terminal side of the hydrophobicsequence. Such a constellation is characteristic of typeII transmembrane proteins that are oriented with theirN-terminal region to the cytoplasmic phase of or-ganelle membranes (Levy, 1996; Hartman et al., 1998).Golvesin was not extracted at high pH or ionicstrength conditions, which remove peripheral mem-brane proteins. Only a small fraction of golvesin wassolubilized with 1% Triton X–100, suggesting that thehydrophobic and the positively charged motif cooper-ate in membrane association (figure 1F).

Knockout of the golvesin gene made Dictyosteliumcells more resistant to the anti-tumour agent cisplatin(Li et al., 2000), which suggests a role of golvesin indrug uptake. On the other hand, inactivation of thegene did not reveal a function of golvesin that might beessential for growth and development under standardlaboratory conditions (Schneider, 1999). Therefore,dominant negative effects of overexpressed GFP-constructs are unlikely to affect basic cellular functions.The absence of obvious alterations in cells showingbrilliantly labelled organelles as shown in figure 10,

makes golvesin–GFP constructs excellent markers ofprotein transport pathways in Dictyostelium, and possi-bly other organisms.

4.2. Shape changes of the Golgi complex inDictyostelium visualized by golvesin(C)–GFP

In its compact state, the GA of Dictyostelium re-sembles the GA of mammalian cells rather than thepunctate appearance of the GA in living yeast, visual-ized by GFP-tagged Golgi resident membrane proteins(Wooding and Pelham, 1998). Within fractions of aminute, the GA can change in Dictyostelium cells fromthe compact to a dispersed state by tubulation. Thesefast variations in Golgi organization might correlatewith alternate phases of high and low transport activi-ties. With respect to tubulation or vesiculation alongmicrotubules, the GA of Dictyostelium resembles theGA in mammalian cells as observed in vivo (Cooper etal., 1990; Weidman et al., 1993; Sciaky et al., 1997;Toomre et al., 1999) and in vitro (Cluett et al., 1993;Banta et al., 1995).

The alternation of Golgi extension and compactionsuggests frequent switches between plus- and minus-end directed motor activities (figure 4A). These data areconsistent with the presence in Dictyostelium of mul-tiple kinesins (Pollock et al., 1999), and of cytoplasmicdynein represented by a single heavy chain (Koonce etal., 1992). The peak velocities of Golgi vesicles, prob-ably representing the tips of tubules during protrusionor retraction, were in the range of 3–4 µm·s–1 in bothdirections (figure 5). This is about one order of magni-tude higher than reported from other cells (Ho et al.,1989); Sciaky, et al., 1997; (Toomre et al., 1999). Sincethe half-width of the velocity peaks was in the order of1 s, these high velocities were only detectable when thesampling intervals were short. These data are consis-tent with the high velocities of microtubule movementsupported by a kinesin-like motor from Dictyosteliumin vitro (McCaffrey and Vale, 1989).

As in mammalian cells (Rabouille and Warren, 1997;Lowe et al., 1998; Lowe et al., 2000; reviewed byNelson, 2000), the GA disassembles in Dictyosteliumduring mitosis (Zhu et al., 1993). Time-series analysisusing golvesin(C)–GFP as a marker revealed the fol-lowing features in dividing Dictyostelium cells: (1) dis-assembly of the GA starts by plus-end directed vesiclemovement along microtubules, which subsequentlydepolymerise (Schneider et al., 1999); (2) during mito-sis the Golgi vesicles become uniformly dispersed andremain visible during meta- and telophase; (3) reas-sembly of the GA is assisted by intense centrosomemovements in post-mitotic cells (Neujahr et al., 1998),whereby the aster microtubules act as dust brushes incollecting the dispersed vesicles (figure 7). The spindledisintegrates beforehand and therefore cannot partici-pate in the partitioning of Golgi vesicles to the daugh



ter cells, except that some vesicles may adhere torudiments of the spindle that are retained after mitosis(figure 7, frames 367-s and 463-s). Since the contractilevacuole network is reconstituted from vesicles at thesame stage but in a different way (Gabriel et al., 1999),it appears that selective Golgi vesicles are activated atpost-mitosis to attach to microtubules.

4.3. GFP–(N)golvesin as a marker to monitorendosome processing

At the beginning of phagocytosis or of fluid-phaseuptake by macropinocytosis, actin filaments assemblearound the phagocytic cup together with coronin andAIP1, two actin-binding proteins of the WD40-repeatfamily that enhance phagocytosis (Maniak et al., 1995;Konzok et al., 1999). After dissociation of these proteinsfrom the endosome membrane, golvesin-containingvesicles begin to interact with the endosomes, resultingin their decoration with GFP–(N)golvesin until theprotein is retrieved prior to exocytosis (figure 8A–J).Late endosomes depleted of GFP–(N)golvesin aredecorated with two isoforms of vacuolin and againwith actin and coronin (Rauchenberger et al., 1997).Thus the GFP-tagged golvesin fills a gap in the list ofmembrane-associated proteins to be used in Dictyoste-lium as stage-specific markers for endosomes in vivo.The association of golvesin with endosome membranesappears to parallel the integration of vacuolar H+-ATPase into these membranes, i.e. the proton pumpresponsible for acidification of the endosomes (Marga-ret Clarke, Oklahoma Medical Research Foundation,personal communication).

Dictyostelium cells contain a second major post-Golgicompartment, the contractile vacuole (CV) network,which is separated from the endosomal system (Gab-riel et al., 1999). Golvesin labelled the CV system, butless than the endosomes (figure 8N). On the contrary,vacuolar H+-ATPase is about 10-fold enriched at CVmembranes as compared to endosomes (Fok et al.,1993), and a GFP-fusion of dajumin is specificallylocated to the CV membranes (Gabriel et al., 1999).

In summary, we have introduced golvesin fusionscarrying GFP at one or the other end of the polypep-tide chain as distinct markers to study organelle dy-namics in live Dictyostelium cells. Since a GFP tag at theC-terminal end of golvesin acted as a specific blockerof translocation to any post-Golgi compartment, thisconstruct proved to be a highly specific Golgi markerwith only a low background in the ER. Alternatively,the strong labelling of endosomes with N-terminallytagged golvesin made this construct useful as a markerfor post-Golgi vesicle processing. These data show thata GFP tag placed to the right end of a protein can beused for blocking protein transport at a defined step ofthe trafficking pathway.

Acknowledgments. We thank Widmar Tanner, Re-gensburg, for yeast mutants, and colleagues of theMax-Planck-Institut für Biochemie, Martinsried, for as-sistance: Monika Westphal for vectors and cells pro-ducing GFP–α-tubulin, Markus Maniak for discussionsand antibodies, Richard Albrecht for establishing sub-second fluorescence imaging, Igor Weber for imageorganization, and Gerard Marriott for the �-tubulinantibodies. The work was supported by the DeutscheForschungsgemeinschaft (SFB266/D7), and the Fonds derChemischen Industrie.

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Golvesin-GFP fusions as distinct markers for Golgi and post-Golgi vesicles in Dictyostelium cells

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