Microtubule depolymerization inhibits transport of cathepsin D from the

Golgi apparatus to lysosomes

JOCHEN SCHEEL, RAFFAELE MATTEONI*, THOMAS LUDWIG, BERNAKD HOFLACK and

THOMAS E. KREIS

European Molecular Biology Laboratory, Meyerhofstrasse 1, D-6900 Heidelberg, FRG

•Present address: ENICHEM Laboratories, Via Ramarini 32,1-00015 Monterotondo (Rome), Italy

Summary

Lysosomes as well as a prelysosomal compartmentrich in the mannose 6-phosphate receptor are clus-tered close to the Golgi apparatus in the perinuclearregion of the microtubule organizing center ininterphase human skin fibroblasts. The spatial or-ganization of these organelles depends on an intactmicrotubule network. Depolymerization of themicrotubules by treatment of cells with nocodazoleleads to random scattering of Golgi elements, theprelysosomal compartment, and lysosomes through-out the cytoplasm. To test whether microtubules andthe spatial organization of these organelles are im-portant for efficient transport of lysosomal enzymes,the effect of microtubule depolymerization on thematuration of newly synthesized cathepsin D wasstudied. An up to fivefold inhibition of proteolyticmaturation of cathepsin D was observed in drug-

treated cells. This effect was due to a decreased rateof transport of cathepsin D from the Golgi apparatusto lysosomes. Depolymerization of microtubules didnot inhibit transport of cathepsin D from the endo-plasmic reticulum to the trans-Golgi network. Fur-thermore, synthesis of the phosphomannosyl markerpresent on cathepsin D was not affected by nocod-azole. These results suggest that efficient transport ofcathepsin D from the Golgi apparatus to a prelysoso-mal compartment and lysosomes is facilitated bymicrotubules and the spatial organization of theseorganelles.

Key words: transport of cathepsin D, Golgi apparatus,lysosomes, microtubules.

Introduction

The microtubule network plays a central role in theorganization of the cytoplasm and in membrane traffic inanimal cells (for reviews, see Schliwa, 1984; Vale, 1987).Movement of Golgi elements, endosomes and lysosomesalong microtubules has been characterized in varioussystems (Herman and Albertini, 1984; Matteoni andKreis, 1987; DeBrabander etal. 1988; Ho etal. 1989). Inaddition, the localization of most of the cytoplasmic organ-elles, including the endoplasmic reticulum, the Golgiapparatus and lysosomes, depends on microtubules(Kupfer etal. 1982; Terasaki etal. 1986; Matteoni andKreis, 1987; Swanson et al. 1987; Heuser, 1989). The Golgiapparatus, a prelysosomal compartment rich in the cation-independent mannose 6-phosphate (Man-6-P) receptor andlysosomes are arranged in the perinuclear region of themicrotubule organizing center (for reviews, see Thybergand Moskalewski, 1985; Hopkins, 1986; Singer andKupfer, 1986; Kreis, 1990; Griffiths et al. 1988, 1990). Ithas been speculated that this spatial apposition of theGolgi complex and endocytic organelles could facilitateintercompartmental transport (Pastan and Willingham,1985; Kreis, 1990).

Vesicular traffic between the Golgi apparatus and theendocytic compartment occurs in both directions. Newlysynthesized lysosomal enzymes are sorted via interactionJournal of Cell Science 96, 711-720 (1990)Printed in Great Britain © The Company of Biologists Limited 1990

with Man-6-P receptors in the Golgi apparatus and trans-ported to a prelysosomal compartment before final deliv-ery to lysosomes (for reviews, see von Figura and Hasilik,1986; Kornfeld, 1987; Storrie, 1988; Kornfeld and Mell-man, 1989). On the other hand, recycling membraneproteins like the Man-6-P receptors or the transferrinreceptor are transported from endosomes to the Golgiapparatus (Snider and Rogers, 1985; Duncan and Korn-feld, 1988; Goda and Pfeffer, 1988). In addition LEP100, alysosomal membrane glycoprotein, shuttles between lyso-somes and endosomes (Lippincott-Schwartz and Fam-brough, 1987).

The lysosomal hydrolase cathepsin D is synthesized inhuman skin fibroblasts as a precursor of Mr 53 000 carry-ing two N-linked oligoaaccharides (Hasilik and Neufeld,1980a; Hasilik and von Figura, 1981). During transportthrough the Golgi apparatus most of this precursor ac-quires Man-6-P residues (Hasilik and Neufeld, 19806),which serve as the sorting signal for delivery to thelysosomes (Kornfeld and Mellman, 1989). This precursor isthen processed, most likely in a prelysosomal compart-ment, to an intermediate form of MT 47 000 (Gieselmannet al. 1983; Braulke et al. 1988). The precise characteriz-ation of this prelysosomal compartment and its locationhave not yet been established. It has been suggested,however, that it is identical to the late endosomal compart-ment that is rich in the cation-independent Man-6-P

711

receptor (Griffiths et al. 1988, 1990; see also Geuze et al.1988). Further processing of cathepsin D to the matureform (Mr 31000 and Mr 14000) occurs in lysosomes(Gieselmann et al. 1983,1985). We have used these differ-ent forms of cathepsin D as hallmarks of the lysosomalpathway to test the hypothesis that microtubules and themicrotubule-dependent organization of the organelles in-volved in trafficking of lysosomal enzymes are essentialfor efficient intercompartmental transport.

Materials and methods

Cell culture and drug treatment of cellsHuman skin fibroblasts were grown in O--MEM supplementedwith 10 % fetal calf serum, 2 mM glutamine and 1 % penicillin andstreptomycin under standard culture conditions. Unless other-wise indicated, microtubules were depolymerized by incubatingcells for l h at 37 °C in culture medium containing 10 JJM nocod-azole (Sigma Chemie GmbH, Deisenhofen, FRG). In some exper-iments we used lower concentrations of nocodazole, or 10;<Mcolchicine (Calbiochem GmbH, Frankfurt, FRG) for 1 h at 37 °C inculture medium.

Indirect immunofluorescenceCells grown on glass coverslips were fixed: (1) in 3% formal-dehyde and 0.02% glutaraldehyde in PBS for 15min at roomtemperature and permeabilized with 0.1% Triton in PBS for4min; or (2) for 4min in methanol at —20°C after pre-extractionwith detergent to remove soluble protein as described (Kreis,1987). The Golgi apparatus was labeled with rabbit antibodiesagainst galactosyltransferase (Roth and Berger, 1982). A murinemonoclonal antibody against a lysosomal glycoprotein, hLAMP-2(Mane et al. 1989), was used to label the lysosomes. The cation-independent Man-6-P receptor was labeled using a rabbit serumagainst the bovine receptor (Griffiths etal. 1988). Microtubuleswere labeled with a murine monoclonal antibody (1A2) against a-tubulin (Kreis, 1987). Incubation with fluorescently labeled sec-ondary antibodies and fluorescence microscopy was performed asdescribed (Kreis, 1986).

Metabolic labeling of cellsSubconfluent cultures of human skin fibroblasts were incubatedfor 15min in methionine-free culture medium and then labeledwith SOO^Ciml"1 of L-[35S]methionine (Amersham Buchler,Braunschweig, FRG). After 10-20 min cells were washed threetimes in PBS and chased for various periods of time in mediumcontaining 1 mM methionine. A 2 ml sample of chase medium wasadded to a 60 mm dish for immunoprecipitation of cathepsin Dsecreted into the medium. Cells were either scraped into PBS (forsubcellular fractionation) or solubilized with 0.5 ml of 1 % Tritonin 50 mM NaCl, 50 mM Tris-HCl, lnu i EDTA, lmgrnl"1 bovineserum albumin (BSA), pH 7.4, per 60 mm dish.

Immunoprecipitation of cathepsin DCell lysates, media, subcellular fractions and fractions from theMan-6-P receptor affinity column were adjusted to 1 % Triton X-100,150 mMNaCl, 50 mM Tris-HCl, lmM EDTA, l m g m P 1 BSA,pH7.4. Samples were centrifuged at 15 000 # for 10 min at 4°Cand 0.1vol. of protein A-Sepharose (Pharmacia LKB GmbH,Freiburg, FRG) was added to the supernatants. These suspen-sions were incubated on a shaker for 1 h at 4°C and the Sepharosewith unspecifically bound material was removed by centrifu-gation as described above, and then 0.01 vol. of a rabbit antiserumagainst cathepsin D (Ludwig et al. unpublished data) was addedto the supernatants and these mixtures were incubated overnightat 4°C. Immunocomplexes were sedimented with 0.1vol. of pro-tein A-Sepharose after incubation for 1 h at 4°C by centrifugationat 5000g for 2 min at 4°C. Protein A-Sepharose pellets werewashed seven times with 0.5% Triton X-100, 0.4 M NaCl, 50 mMTris-HCl, pH7.4, and once with 50 mM Tris-HCl, pH7.4. Final

pellets were boiled in 60 fd gel sample buffer and immunoprecipi-tated material was separated by polyacrylamide gel electrophor-esis in the presence of sodium dodecyl sulfate (SDS-PAGE) in 9 %polyacrylamide gels according to Laemmli (1970). Gels wereincubated in 1 M sodium salicylate for 20 min (Chamberlain,1979), dried and fluorographs were taken on preflashed KODAKX-OMAT AR films (Eastman Kodak, Rochester, NY) (3-21 daysat —70°C). Radioactivity associated with the different forms ofcathepsin D was quantified by densitometric scanning of thefluorographs (Quick scan R&D, Desaga, Heidelberg, FRG).

Digestion of immunoprecipitated material withendoglycosidase HImmunoprecipitated material bound to protein A-Sepharose wasboiled with 30 (A of 1% SDS, 200 mM sodium citrate, pH5.5.Samples were cooled after 4 min and 120 /d of 200 mM sodiumcitrate, pH5.5, containing 2.5jugml"1 pepstatin was added.Samples were split into two aliquots, one of which receivedendoglycosidase H (50munitsml~ final concentration) (Seika-gaku, Tokyo, Japan). Samples with and without endoglycosidaseH were incubated for 18 h at 37 °C and then analyzed bySDS-PAGE.

Subcellular fractionation on Percoll gradientsLabeled fibroblasts (from 15 cm Petri dishes) were scraped into5 ml of PBS using a rubber policeman. Cells were sedimented at1000 gfor 5 min at 4°C and resuspended in 1 ml of 250 mM sucrose,2 mM EDTA, 3 mM imidazole, pH 7.2. Cells were homogenized byfive strokes in a tight-fitting Dounce homogenizer (WheatonScientific, Millville, USA). A postnuclear supernatant was pre-pared by centrifugation of the homogenate at 800 g for 10 min at4°C, loaded over 8 ml of 27 % Percoll (Pharmacia LKB GmbHFreiburg, FRG) in 250 mM sucrose, 3mM imidazole, lmgmlBSA, pH 7.2, on a 0.5 ml cushion of 2 M sucrose and sedimented at20000gVnOX for 2h at 4°C in a Ti50 rotor (Beckman Instruments,Inc., Fullerton, CA). Fractions of 0.6 ml were collected from thetop and assayed for density (Pertoft etal. 1978), radioactivity,/3-hexosaminidase activity (Pool et al. 1983) and galactosyltrans-ferase activity (Strous and Berger, 1982).

Mannose 6-phosphate receptor affinity columnCation-independent Man-6-P receptor purified from bovine liverwas coupled to Affigel 10 (Bio-Rad Laboratories GmbH, Muen-chen, FRG) as described elsewhere (Varki and Kornfeld, 1982;Hoflack etal. 1987). Cell lysate (0.5ml) prepared as describedabove was supplemented with 5mM /3-glycerophosphate andapplied to a 3 ml Man-6-P receptor affinity column equilibrated inCB (50 mM imidazole, 150 mM NaCl, 0.05% Triton X-100, 0.1 mgBSA ml"1, 5mM /J-glycerophosphate, pH7.0). The column waswashed with CB after 2 h of incubation at 4°C and fractions of 2 mlwere collected. Fractions containing radioactivity were pooled.Lysosomal enzymes bound to the receptor were eluted with 5 mMMan-6-P in CB and fractions containing radioactivity werepooled.

Results

Role of microtubules in the spatial organization ofendocytic organelles and the Golgi apparatusThe Golgi apparatus, the prelysosomal compartment richin the cation-independent Man-6-P receptor, and lyso-somes were labeled in human skin fibroblasts with anti-bodies against galactosyltransferase (Roth and Berger,1982), the cation-independent Man-6-P receptor (Griffithset al. 1988), and hLAMP2 (Mane et al. 1989), respectively.It has been proposed that the compartment containing thebulk of the Man-6-P receptor is a prelysosomal compart-ment where lysosomal enzymes are released from thereceptor (Griffiths etal. 1988). Cathepsin D colocalizedwith hLAMP2 by immunofluorescence (not shown). All

712 J. Scheel et al.

three compartments were clustered in the perinuclearregion of the microtubule organizing center (Fig. 1). Thethree compartments were distinct, but the Golgi appar-atus and the prelysosomal compartment rich in the Man-6-P receptor were closely apposed (see also Griffiths et al.1988, 1990).

Depolymerization of microtubules by treatment of thefibroblasts for l h at 37 °C with IOJM nocodazole resultedin random scattering of the Golgi apparatus, the prelyso-somal compartment rich in the Man-6-P receptor, andlysosomes throughout the cytoplasm (Fig. 1). Thus, theclose apposition of these organelles in the perinuclearregion requires an intact microtubule network.

Treatment of cells with either 10 fiM nocodazole or 10 UMcolchicine during 1 h at 37 °C led to the complete depolym-erization of microtubules (Fig. 2C,E) and dispersal of theGolgi apparatus (Fig. 2D,F). Treatment of cells with0.3 /JM nocodazole for up to 4h did not completely depolym-erize microtubules (see also Kreis, 1987) but led to somedispersal of Golgi elements (Fig. 2G,H). Scattering of theprelysosomal compartment rich in the Man-6-P receptor,and lysosomes, was comparable to dispersal of the Golgiapparatus. On the other hand, 0.1 ^M nocodazole inducedminor changes in the microtubule network (compareFig. 2A and I) and the Golgi apparatus remained appar-ently normal (compare Fig. 2B and J). Treatment of cellswith 10 fJM nocodazole for 1 h at 37 °C had no significanteffect on the morphology of the endoplasmic reticulumvisualized by immunofluorescence labeling with anti-bodies against protein disulfide isomerase (data notshown).

Effect of depolymerization of microtubules on thematuration of cathepsin DThree major forms of cathepsin D can be distinguishedduring maturation of this lysosomal hydrolase. A precur-sor form of Afr53000, an intermediate form of Afr47000,and the mature heterodimeric form consisting of aMr 31000 heavy and a Afr14 000 light chain are predomi-nantly present in the endoplasmic reticulum and the Golgiapparatus, a prelysosomal compartment, and the lyso-somes, respectively (Gieselmann et al. 1983; Braulke et al.1988). A small fraction of the 53 000Mr precursor form(<10%) is secreted into the medium of cultured cells(Hasilik and Neufeld, 1980a). In this study 'cathepsin D'refers to all three forms of the protein.

Maturation of cathepsin D was followed by metaboliclabeling of human skin fibroblasts with [35S]methionine.Cells were pulsed with SOO^Ciml"1 for 15min at 37 °Cand chased for various periods of time. Labeled cathepsinD was immunoprecipitated both from the cell lysates andthe corresponding cell culture medium with specific anti-bodies and analyzed by polyacrylamide gel electrophor-esis. Processing of cathepsin D from precursor to inter-mediate and mature forms was followed in control andnocodazole-treated cells (Fig. 3), as well as in cells treatedwith colchicine or low concentrations of nocodazole(Fig. 4). The rate of processing of cathepsin D to theintermediate and mature forms was reduced up to fivefoldin cells without microtubules (Fig. 3). A total of 54% oflabeled cathepsin D was present in the precursor formafter 2.5 h of chase in nocodazole-treated cells, whereasonly 9 % was found in the control cells (Fig. 3B). Theintermediate form reached its highest concentration after90-120 min of chase in control cells, but only after 3h inthe nocodazole-treated cells (Fig. 3B). After 2.5 h of chase71 % of total cathepsin D has been processed to the mature

form (only the Mr31000 form was analyzed here) incontrol cells, compared to 13 % in nocodazole-treated cells(Fig. 3B). Complete processing to the mature form oc-curred also in nocodazole-treated cells after long periods ofchase (9h; Fig. 3).

We observed no differences in the inhibition of process-ing of cathepsin D after 2 and 3 h of chase when micro-tubules were completely depolymerized with either nocod-azole or colchicine (Fig. 4). Treatment of cells with lowdoses of nocodazole (0.1 UM, see also Fig. 2), however, hadno effect on the processing of cathepsin D. These resultssuggest that reduced rates of maturation of cathepsin Dare probably due to depolymerization of microtubules,rather than non-specific effects of nocodazole.

About 5 % of the precursor was secreted in both controland nocodazole-treated cells after 9 h of chase. Secretion ofcathepsin D, however, was significantly delayed in thenocodazole-treated cells (Fig. 3). Similar effects of micro-tubule depolymerization on the kinetics of transport ofvesicular stomatitis virus glycoprotein from the trans-Golgi network to the cell surface have been measured (W.C. Ho and T. E. Kreis, unpublished results).

Analysis of transport of cathepsin D to lysosomes by cellfractionationTo test whether the inhibitory effect of nocodazole onprocessing of cathepsin D was due to inhibition of trans-port, fibroblasts labeled with [36S]methionine were hom-ogenized and fractionated by centrifugation through aPercoll gradient. Control or nocodazole-treated cells werepulsed with [^SJmethionine for 15 min and subsequentlychased for 30 min, 1 h and 6 h, respectively, before hom-ogenization. Postnuclear supernatants were loaded on topof a 27 % Percoll gradient and centrifuged as described inMaterials and methods. Fractions were collected andanalyzed for radioactivity, /S-hexosaminidase and galac-tosyltransferase activity (Fig. 5). The light fractions (2-6)containing the bulk of the Golgi apparatus and theprelysosomal compartment rich in the Man-6-P receptor(Griffiths et al. 1990), the dense fractions (12-16) enrichedin lysosomes, and the intermediate fractions (7-11), werepooled as indicated (Fig. 5A). Cathepsin D was immuno-precipitated from these three pools (Fig. 5B).

In control cells, cathepsin D immunoprecipitated fromthe pool of the light fractions was 96 %, 70 % and 4 % oftotal cathepsin D after periods of chase of 30 min, 1 h and6h, respectively (Fig. 5B). Cathepsin D immunoprecipi-tated from the pooled heavy fractions was 2 %, 27 % and95 %, after 30 min, 1 h and 6 h, respectively (Fig. 5B). Onlyprecursor and intermediate forms of cathepsin D werefound in the pooled light fractions, whereas the pooledheavy fractions contained only the intermediate and ma-ture forms of cathepsin D. Thus, we find that the proteo-lytic cleavage steps during maturation of cathepsin D andits transport to the fraction containing the lysosomes arein good agreement with previous findings reported byGieselmann et al. (1983). Virtually no difference in theamounts of cathepsin D immunoprecipitated from thelight and heavy fractions could be detected in control andnocodazole-treated cells after 30 min and 6h of chase(Fig. 5B). This result suggests that sorting and targetingof cathepsin D is unaffected by the nocodazole treatment ofthe cells. At the intermediate chase period (1 h), however,only 5 % of the total cathepsin D could be immunoprecipi-tated from the pooled heavy fractions in the nocodazole-treated cells (Fig. 5B). Therefore, transport of cathepsin Dto lysosomes was significantly delayed in the absence of

Microtubule-dependent transport of cathepsin D 713

Fig. 1. The organization of the Golgi apparatus, the Man-6-P receptor compartment, and lysosome clusters in the perinuclear regionof human skin fibroblasts depends on microtubules. The Golgi apparatus (A,C), the prelysosomal compartment rich in the Man-6-Preceptor (E,G), and lysosomes (B,D,F,H) were visualized by double immunofluorescence using specific antibodies againstgalactosyltransferase, the cation-independent Man-6-P receptor, and hLAMP-2, respectively. Identical cells are shown in A,B; C,D;E,F; G,H. Treatment of cells with nocodazole for l h at 37 °C induced scattering of the three compartments throughout the cytoplasm(C,D,G,H). The positions of distinct Golgi elements are indicated by arrowheads in C and D. Bar (A-H), 20 /an.

714 J. Scheel et al.

Fig. 2. Depolymerization of microtubules and scattering of Golgi elements. Human skin fibroblasts were incubated without drugs(A,B)i with 10/iM nocodazole (CJ)) or 10/JM colchicine for l h (E,F), and 0.3/an (G,H) or 0 . 1 / M nocodazole for 4h (I,J). Cells were pre-extracted to remove soluble cytoplasmic protein, fixed and labeled with an antibody against tubulin (A,C,E,G,I). Sincegalactosyltransferase was also extracted under these conditions, cells treated identically with microtubule-disrupting drugs werefixed directly and labeled with antibodies against galactosyltransferase (B,D,F,H,J). Bar (A-J), 20/an.

Microtubule-dependent transport of cathepsin D 715

microtubules, when Golgi elements, prelysosomal com-partment and lysosomes were scattered throughout thecytoplasm. In the first 60min of chase this delay wasequivalent to an inhibition of about fivefold.

Depolymerization of microtubules does not inhibittransport of cathepsin D to the Golgi apparatusA fraction of the two oligosaccharide side-chains of thecathepsin D precursor (Mr 53 000) is converted in the Golgiapparatus to endoglycosidase H-resistant forms (Hasilik

and von Figura, 1981). Digestion of metabolically labeledcathepsin D precursor with endoglycosidase H was used tomeasure its transport to the Golgi apparatus (Fig. 6).Control cells and cells treated with nocodazole werelabeled for 10 min with [^SJmethionine and chased for upto 60 min. Cathepsin D was immunoprecipitated and halfof the precipitated material was digested with endogly-cosidase H as described in Materials and methods. About20 % of the precursor acquired (partial or complete) endo-glycosidase H resistance after 60 min of chase (Fig. 6). Nosignificant difference in the rate and extent of acquisition

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Fig. 3. Effect of microtubule depolymerization by nocodazole on the rate of maturation of cathepsin D. Fibroblasts were labeled with[^Jmethionine for 15 min and chased for up to 9 h. A. Cathepsin D was immunoprecipitated from cell lysates (c) and culture media(m) of control and nocodazole-treated cells after various periods of chase and analyzed by SDS-PAGE and fluorography. Thepositions of the precursor (Mr53000), intermediate (M,. 47 000) and mature (Mr31000) forms of cathepsin D are indicated (top tobottom). Only the Afr53 000 precursor form was found secreted in the culture medium. B. Radioactivity associated with the differentforms of cathepsin D in the cell lysates and the culture medium separated by SDS-PAGE was quantified by densitometric scanningof the fluorographs. The percentages of total cathepsin D (53000, 47000 and 31000 Mr bands) for each form of cathepsin D werecalculated. (D D) Control cells; (A • ) nocodazole-treated cells.

716 J. Scheel et al.

Fig. 4. Depolymerization of microtubules inhibits maturation N0C0(JaZ0l6of cathepsin D. Fibroblasts were labeled with [^SJmethioninefor 15 min and chased for 2 or 3 h with or without the drugs _ .indicated. Cathepsin D was immunoprecipitated from the cell UOlCnlCinelysates and analyzed by SDS-PAGE and fluorography asdescribed in the legend to Fig. 3.

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of endoglycosidase H resistance could be detected betweencontrol and nocodazole-treated cells (Fig. 6). This suggeststhat transport of cathepsin D from the endoplasmic reticu-lum to the Golgi apparatus is not affected by nocodazole.

Synthesis of the phosphomannosyl marker is not affectedby treatment of cells with nocodazoleBinding of lysosomal enzymes to Man-6-P receptors allowsthe segregation in the trans-Golgi network of these pro-teins routed to lysosomes from those destined to besecreted (for reviews, see von Figura and Hasilik, 1986;Kornfeld, 1987; Kornfeld and Mellman, 1989). CathepsinD acquires the Man-6-P recognition signal in a two-stepprocess, which occurs in a pre-Golgi compartment and thecis-cisternae of the Golgi apparatus (for a review, seeKornfeld and Mellman, 1989). To confirm that phosphoryl-ation of mannose residues was not affected by the nocod-azole treatment of cells, binding of cathepsin D to thecation-independent Man-6-P receptor was analyzed(Fig. 7). Fibroblasts with intact or depolymerized micro-tubules were pulsed for 10 min with [ S]methionine andchased for 46 min to allow transport of the cathepsin Dprecursor to the Golgi apparatus. Cell lysates were pre-

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Fig. 5. Transport of cathepsin D to lysosomes. Fibroblasts werelabeled with [™S]methionine for 20 min and chased for 30 min,l h and 6h, respectively. Postnuclear supernatants (PNS) wereprepared and fractionated on Percoll gradients (as described inMaterials and methods). Fractions of 0.6 ml were collected fromthe top of the gradient. A. Fractions were analyzed for density,radioactivity, /J-hexosaminidase activity and galactosyl-transferase activity. A. The profile of a gradient after 1 h ofchase. B. Fractions 2-6 containing the Golgi membranes aswell as late endosomes, the intermediate fractions 7-11, andfractions 12-16 containing lysosomes were pooled as indicatedin A. Cathepsin D was immunoprecipitated from these pools,and analyzed by SDS-PAGE and fluorography. The three formsof cathepsin D are indicated on the right as described in thelegend to Fig. 3.

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Fig. 6. Digestion of cathepsin Dwith endoglycosidase H. Fibroblasts

_j_ were labeled for 10 min with[^Slmethionine, chased for up to60 min, and cathepsin D wasimmunoprecipitated from lysates of

^ ^ control and nocodazole-treated cells.Immunoprecipitation andendoglycosidase H treatment wereperformed at the time pointsindicated as described in Materialsand methods. The following threeforms of endoglycosidase H-digestedcathepsin D precursors could bedistinguished with MT of 53 000(both oligosaccharide chainsresistant), about 50 000 (one

6 0 oligosaccharide chain resistant), andabout 47 000 (both oligosaccharidechains sensitive).

pared and fractionated on a Man-6-P receptor affinitycolumn as described in Materials and methods. No signifi-cant difference in binding of cathepsin D precursor to theMan-6-P receptor affinity column was measured withlysates from control or nocodazole-treated cells (58%versus 57 %; Fig. 7). Since binding of cathepsin D to theMan-6-P receptor was also not modified by the nocodazoletreatment, we concluded that inhibition of transport tolysosomes was not due to alterations in the state ofphosphorylation of cathepsin D by the drug. Therefore,nocodazole does not affect the sorting of lysosomal en-zymes by the Man-6-P receptors. This is also consistentwith the pulse-chase experiment showing that secretionof cathepsin D was not increased in nocodazole-treatedcells (Fig. 3).

Discussion

Microtubule-depolymerizing agents have been instrumen-tal in analyzing the involvement of microtubules in theprocesses of intracellular transport (see, for example,Freed and Lebowitz, 1970; Herman and Albertini, 1984;Phaire-Washington et al. 1980; DeBrabander et al. 1988;Ho etal. 1989; Kreis, 1990). They not only disrupt thetracks along which movement of vesicles proceeds, butthey also have profound effects on the spatial organizationof cytoplasmic organelles; for example, both elements ofthe Golgi apparatus and lysosomes are scattered randomlythroughout the cytoplasm in the absence of an intactmicrotubule network (Kupfer etal. 1982; Matteoni andKreis, 1987; Swanson et al. 1987; Heuser, 1989). We have

Control Nocodazole

uFig. 7. Binding of cathepsin D to the cation-independent Man-6-P receptor. Fibroblasts were labeled with [^Slmethionine for10 min and chased for 45 min. Cell lysates prepared fromcontrol and nocodazole-treated cells were fractionated on aMan-6-P receptor affinity column as described in Materials andmethods. Cathepsin D (precursor form, Mr53 000) was immuno-precipitated from pools of the fractions containing unbound(u; flow through) and bound (b) material.

used the maturation of the lysosomal hydrolase, cathepsinD, as a model in which to study whether microtubules andthe microtubule-dependent spatial organization of theGolgi apparatus and lysosomes are important for efficienttransport between these two organelles.

The rate of transport of cathepsin D from the Golgiapparatus to lysosomes was reduced up to fivefold in theabsence of microtubules as monitored by the proteolyticprocessing of cathepsin D and delivery of cathepsin D fromthe Golgi apparatus (light membrane fraction) to lyso-somes (heavy membrane fraction) followed by cell frac-tionation. Comparison of the kinetics of processing ofcathepsin D in control and nocodazole-treated cellssuggests that: (1) transport of cathepsin D from the trans-Golgi network to the compartment where processing to theintermediate form occurs was inhibited significantly innocodazole-treated cells; and (2) transport from this prely-sosomal compartment to lysosomes was inhibited to asmaller extent. Neither transport from the endoplasmicreticulum to the Golgi apparatus, nor sorting of cathepsinD was affected by nocodazole (see Figs 5 and 6). Further-more, it has been shown previously that formation ofcoated vesicles at the plasma membrane remains unaffec-ted by the treatment of cells with nocodazole (Gruenberget al. 1989).

Inhibition of transport of cathepsin D by depolymeriz-ation of microtubules could be due to the disappearance ofthe microtubule tracks along which movement of vesiclesmight occur, or to the disruption of the spatial arrange-ment of the organelles involved. It is assumed, however,that microtubules are not involved in short-range vesicu-lar transport like that from the endoplasmic reticulum tothe Golgi apparatus, in between Golgi cisternae, or fromthe plasma membrane to early endosomes (see, forexample, Kelly, 1990). We have shown here that, nor-mally, the Golgi apparatus, the prelysosomal compart-ment that is rich in the Man-6-P receptor, and theclustered lysosomes are closely apposed. Depolymerizationof microtubules results in random dispersal of theseorganelles throughout the cytoplasm. Thus, the inhibitoryeffect of nocodazole and colchicine on transport of cathep-sin D to lysosomes may to a large extent be due to thedramatic changes in the spatial organization of theseorganelles. It is conceivable that the nearest neighbourdistances between the organelles are increased upon scat-tering, since these same organelles are randomly dis-persed in a much greater volume than initially. In con-trast, the endoplasmic reticulum remains extended

718 J. Scheel et al.

throughout the cytoplasm and its morphology is notsignificantly changed by treatment with nocodazole forshort periods of time. Therefore, distances between theendoplasmic reticulum and elements of the Golgi appar-atus should, in principle, not alter very much, which isconsistent with our observation that nocodazole does notaffect transport from the endoplasmic reticulum to theGolgi apparatus. Although we cannot exclude the possi-bility that vesicular transport from the Golgi apparatus tolysosomes occurs along microtubules, we consider it morelikely that it is the microtubule-dependent apposition ofGolgi apparatus, prelysosomal compartment that is rich inthe Man-6-P receptor, and lysosomes, in the perinuclearregion that ensures efficient transport of newly syn-thesized cathepsin D to lysosomes.

Evidence has been presented recently that microtubule-independent factors may be involved in the positioning ofthe Golgi apparatus (Turner and Tartakoff, 1989; Ho andKreis, unpublished observations), and perhaps also thelysosomes (Matteoni and Kreis, 1987), in the region of themicrotubule organizing center. Further work will berequired to characterize the molecules that are involved inthe spatial organization of these organelles.

We thank Drs Thomas August, Eric Berger, Steve Fuller andStuart Kornfeld for their generous gift of antibodies, RodrigoBravo for providing the human skin fibroblasts, and GarethGriffiths for critically reading the manuscript.

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