A Chinese Hamster Ovary Cell Line with a Temperature-conditional ...

Vol. 268, No. 34, Iasue of December 5, PP. 25357-25363,1993 Printed in U.S.A.

AL THEJOURNALOF B I O ~ I C CHEMISTRY 0 1993 by The American Society for Rimherniatry and Molecular Biology, Inc.

A Chinese Hamster Ovary Cell Line witha Temperature-conditional Defect in Receptor Recycling Is Pleiotropically Defectivein Lysosome Biogenesis* (Received for publication, July 8, 1993, and in revised form, August20, 1993)

Russell B. Wilson$, Cynthia CorleyMastickO, and RobertF. Murphyn From the Department of Biological Sciences and the Center for Light Microscope Imaging and Biotechnology, Carnegie Mellon University, Pittsburgh, Pennsylvania 15213

We have previously described the isolation of a Chi- ceptor-ligand dissociation, and that within this compartment be recycled back to thecell surface, such as nese hamster ovary cell line, TfTl.ll, that has a pleio- proteins destined to tropic, temperature-conditional defect in receptor recytransferrin, aresegregated from those, such as a,-macroglobucling (Cain,C. C., Wilson, R. B., and Murphy,R. E (1991) lin, that are ultimatelydegraded within lysosomes. J. Biol. Chem. 266,11746-11752).These cells show a rapid After dissociationof ligand-receptor complexes, receptors are loss of cell surface receptors upon temperature shift found due primarily in tubularcomponents of the earlyendosomal to a reduction in the rate of receptor recycling. We show membrane, while ligands and integral membrane proteins that herethat, in additiontoalteredreceptorrecycling, are destinedfor delivery to lysosomes are located mainlyin the TfT1.ll cells show three defectsin lysosome biogenesis. vacuolar portion (8).Retention of ligands within the vacuolar At the nonpermissive temperature, they1) redistribute portion may result from surface to volume differences between at least one lysosomal enzyme from lysosomes to endo- the vacuolar portion and the tubular extensions (9,10). The somes, 2) fail to transfer fluid-phase material from early mechanisms by which receptors are concentrated in the tubufail to accumuendosomes to later compartments, 3) and late fluid-phase markers due to increased efflux of in- lar extensions of the earlyendosome and by which receptor-rich membranes are removed are currently unknown. ternalized material. The results suggest that the proIsolation and characterization of eukaryotic mutants have of cesses of recycling from the endosome and movement provided important insights into membrane traffk (for reviews, materialfromendosomestolysosomesaretightly see Refs. 11 and 12). To further elucidate the mechanisms and linked. identify the factorsinvolved in the recycling steps of receptormediated endocytosis, we have isolated Chinese hamster ovary (CHO)‘ cell lines with temperature-conditional defects in reDuring receptor-mediated endocytosis (see Refs. 1-3), li- ceptor recycling (13). Fluorescence-activated cell sorting of mugands bind to specific receptors on the plasma membrane. at tagenized CHO cells was used to isolate lines that retained, These ligand-receptor complexes are internalized from coated a nonpermissive temperature, a pulse of fluorescently labeled pits by a n invagination of the plasma membrane form to coated transferrin aftera chase in the presence of unlabeled transfervesicles. Coated vesicles rapidly lose their clathrin coat and rin (the lines are given the prefur TfT to denote their isolation fuse with one another and/or with pre-existing structures, re- due to Bansferrin Trapping). The retention of transferrin in sulting in theappearance of internalizedmembraneand one of the lines, TfTl.11, is due toa 5-10-fold reduction in the soluble content ina class of tubulo-vesicular organelles termed initial rateof transferrin recycling and results ina decrease in early endosomes. Present within the early endosomal memthe numberof transferrin receptors present on the cell surface. brane are proton pumps that generatea mildly acidic environ- The defect in TfT1.ll is pleiotropic since a,-macroglobulin rement of approximately pH6 (for reviews, seeRefs. 4 and 5).The ceptors are also lost from the cell surface at the elevated temmoderate pH within early endosomes is regulated, at least in perature (13). part, by the activity of the Na,K-ATPase (6, 7). Since many Because treatments reported to block receptor recycling also ligands dissociate from their receptors at a pH of 6 or below, it block transfer of material to dense compartments and cause a is thought that the earlyendosome plays a critical role in re- redistribution of lysosomal enzymes (14, 15),we examined the fate of internalized fluid-phase material and the distribution of lysosomal enzymes in TfT1.11. We show that after 4 h at the * This work was supported in part by National Institutes of Health nonpennissive temperature,lysosomal enzyme activitywas reGrant GM32508 and NationalScience Foundation Grant DCB-8903657 distributed in Percoll density gradients from dense lysosomes (to R. F. M.). Thecosts of publication of this article were defrayed in part to more buoyant compartments and that fluid-phase material by the payment of page charges. This article must therefore be hereby was not transferred to dense lysosomes. In addition, we show marked “advertisement” inaccordancewith 18 U.S.C.Section1734 that TfT1.llcells are defective in theaccumulation of material solely to indicate this fact. t Lawton Chiles Biotechnology Postdoctoral Fellow of the National internalized by fluid-phase endocytosis and that this defect Institutes of Health supported by Grant GM13179. Present address: results from a n enhanced efflux of internalized material. The Dept. of Pathology and Laboratory Medicine, Tulane UniversityMedi- results suggest that recycling of receptors from endosomes back cal Center, 1430 Wane Ave., New Orleans, LA 70112-2699, to the pbsma membrane and the transfer of material to later 8 Supported by an American predoctoral fellowship of the American linked, andthat a Association of University Women Educational Foundation. Presentad- endocytic compartmentsareintimately dress: Dept. of Signal Transduction, Parke-Davis Pharmaceutical Re- single defect can abrogate both processes. search Division, Warner-Lambert Co., Ann Arbor, MI 48105. ll To whom correspondence should be addressed: Dept. of Biological The abbreviationsused are: CHO, Chinese hamster ovary; a-MEM, Sciences, Camegie Mellon University, 4400 Fifth Ave., Pittsburgh, PA essential minimal medium, a modification, with supplements; XRITC15213. ”el.: 412-268-3480; Fax: 412-268-6571. dextran, substitutedrhodamine isothiocyanate-conjugated dextran.

25357

25358

Receptor Recycling and Lysosome Biogenesis

mixture was removed by aspiration, and the cells were quickly chilled by the additionof 1 ml of ice-cold a-MEM salts. Thecells were washed five times with a-MEM salts, scraped into 0.5 ml of a-MEM salts, and Tissue culture supplies were obtained from Life Technologies Inc. analyzed (all on ice). To determine if the failure of TfT1.ll cells to and other materials were obtained from Sigma unless otherwise noted. from increased efflux of maCells-Tissue culture procedures and growth medium wereas previ- accumulate internalized material resulted a t 4 1"C for 4 hand labeled withXRITCously described (13). The CHO WTB cell line was obtained from Dr. terial, cells were preincubated Brian Stome, and the isolation of the temperature-conditional mutant, dextran for either 5 or 60 min. The cells werechilled and washed with ice-cold a-MEM salts. One set of plates (both WTB and TfT1.ll) was TfT1.11, from CHO WTB has been previously described (13). In all by experiments, cells were plated2-3 days prior to each experiment such maintained at 4 "C while the remaining plates were warmed to"C41 that on the day of the experiment, thecell density was a t most 80% of the addition of warm a-MEM without XRITC-dextran. After various periods of time the cells werewashedwith ice-cold a-MEMsalts, contluency. scraped, and analyzed. To determine the efflux kinetics of material Preparation of Ligands-XRITC-dextran (70,000 daltons) was preinternalized priorto shift to the nonpermissive temperature, cells were pared with the modifications previously described to reduce free dye contamination (16), and transferrin (human diferric, Miles Scientific, incubated at 33 "C for 1 h with XRITC-dextran, washed and either maintained a t 4 "C or incubated at 33 "C for 1 h in the absence of Naperville, IL) was labeled with lZ5I (approximately 1 pCi/pg) using XRITC-dextran. The cells were then either maintained a t 33 "C or inIODO-BEADS (Pierce Chemical Co.) (17). cubated a t 4 1"C for up to 6 h prior to scraping and analysis. Flow Cytometry-A dual laser FACS 440 flow cytometer (Becton Dickinson Immunocytometry Systems) equipped with argonand krypRESULTS ton lasers was used. XRITC fluorescence (568 nm excitation, 100 milThe Density Distribution of Dansferrin Is Unaltered after liwatts) was collected using a 625-nm band-pass filter (35 nm band width). Data was obtained in listmode and analyzed on a VAX 11/750 Temperature Shift-At 41 "C, TfT1.ll recycles endocytosed computer usingConsort/VAX software. A forward scatter threshold was transferrin much more slowly than parental WTB (13).A posused to select events for acquisitionand forward and side scatter gates sible mechanism by which this could occur is the delivery of were used to restrict analysist o single, viable cells. or less buoyant endocytic Subcellular Distribution of Endocytic Markers a n d @-Galactosidase endocytosed transferrin to "later" "To determine the subcellular distributionof transferrin, cells (in 100 compartments such as dense lysosomes. To investigate this mm dishes) were preincubateda t 4 1"C for 4 h or maintained a t 33 "C possibility, the density distribution of endocytosed 1251-Tfwas determined by centrifugation on Percoll gradients. Two types of prior to incubation in serum-free a-MEM containing 1 mg/ml bovine serum albumin and 2 pg/ml lZsI-Tf for 30 min. The cells were then Percoll gradients can be used for fractionating endosomes and washed 5 times with ice-cold a-MEM salts (116 m NaCl, 5.4 m KC], lysosomes. Gradients formed from a starting Percoll concentra0.2 m CaC12,0.8 m MgSO,, 10 m NaH,PO,, pH 7.41, washed once in tion of27% are frequently used to separate endosomes (both HB (250 m sucrose, 10 m Hepes, 2 m EDTA), and scraped with a early and late) from dense lysosomes (20). If a lower starting rubber policeman into HB. Scraped cells were centrifuged a t 1,000 x used, late endosomes are g, for 10 min, resuspended in 4 mlof HB, and homogenized with 10 Percoll concentration (such as 17%) is strokes of a tight-fitting glass Dounce homogenizer (Fisher). The ho- observed to cofractionate with dense lysosomes (20); this bemogenizer was rinsed with 4.5 ml of HB, and the homogenate and rinse havior on 17% Percoll gradients has been confirmed for CHO were combined. The homogenates were then centrifugedfor 10 min at cells (21). The majority of transfenin-containing compartments 1,000 x g, to remove unbroken cells and debris. The postnuclear are normally found in the light peak of both types of gradients. supernatants (7 ml) were mixed with Percoll (Pharmacia LKB BiotechThus, to determine whether transferrin wasbeing abnormally nology Inc., Piscataway, NJ) to a final concentration of 17% in HB (28 ml the distribution of routed to denser compartments inl"1.11, final volume), loaded into Sorvall Ultracrimp tubes (DuPont Instru1251-Tfinternalized during a 30-min incubation was analyzed ments), underlayered with 4 ml of 60%sucrose containing 2m EDTA, and centrifuged for 1 h at 15,750 rpm in a n SV-288 vertical rotor (Du- on 17% Percoll gradients for WTB and TfT1.ll at permissive Pont). Approximately 1-ml fractions werecollected from the top witha and nonpermissive temperatures (Fig. 1). The radiolabeled Buchler Auto Densi-flow IIC (Buchler Instruments, Lenexa, KS). Ra- transferrin was found in the buoyant fractions in both WTB dioactivity present in each fraction was determined by scintillation counting using Ecolume scintillation mixture (ICN, Irvine, CA) and 25 I 1.12 -h expressed a s a percent of the total activity in the gradient. E I 1.10 & To determine the distribution of endocytic compartments accessible I to the fluid-phase marker horseradish peroxidase and to determine the 1.08 2 .distribution of @-galactosidase,approximately 1 x lo7 cellsin two 20 1.06 """" 150-mm dishes were preincubated for 4 h at 33 or 41 "C and then C a 0 incubated for 1.5 h in a-MEM containing 2.5 mg/ml horseradish per1.04 0 Q oxidase (Sigma, Type 11). Cellular organelles were prepared and frac0 tionated on 17 or 27% Percoll density gradients as described above. 15 @-Galactosidaseactivitywasdeterminedineachfractionusing the 4-methylumbelliferyl substrate (18), and horseradish peroxidase activity was determined using o-dianisidine as a substrate (19). Enzyme c activities of each fraction were normalized for the amount of protein r 10 0 loaded onto the gradient and for total enzyme activity present in the c gradient of WTB. Protein concentration was determined using the BCA E a3 protein assay reagent (Pierce) withbovine serum albumin as the stan$ 5 dard. n Analysis of Fluid-phase Endocytosis-For allassays, cells were plated in 12-well tissue culture plates (Corning Glassworks, Corning, N Y ) , preincubated as appropriate, and incubated with mg/ml 2 XRITC0 dextran in a-MEM for the specified time. Mean XRITC-dextran fluo0 5 10 15 20 25 30 rescence was measured byflow cytometry (on ice) and corrected for Fraction number autofluorescence of unlabeled cells before further calculations. Triplicate samples were measured for each condition. FIG. 1.Endocytosed transferrinremains in buoyant endocytic To assess the ability of TfT1.ll cells to accumulate material inter- compartments. WTB (circles) and TfT1.11 (triangles) cells were either nalized by fluid-phase endocytosis, cells were preincubateda t 4 1"C for maintained at 33"C (open symbols) or incubated at 41 "C (closed sym4 h or maintained at 33"C and then incubated with XRITC-dextran for bols) for 4 h prior to labeling with 5 Il/ml 1251-Tf. After a 30-min incuup to 1h before analysis. To determine the kineticsof expression of the bation, postnuclear supernatants were prepared and fractionated on defect in the accumulation of material internalized by fluid-phase en- 17% Percoll density gradients. Fractions were collected from the top. Radioactivity in each fraction were normalized t o the total in each docytosis, cells were preincubateda t 4 1"C for up to 6 h or maintained at 33 "C prior to labeling with XRITC-dextran for 30 min. The labeling gradient. MATERIALSANDMETHODS

--:

=-E 3

Y

and Lysosome Biogenesis

Recycling Receptor

25359

nalized material to dense compartments at the nonpermissive temperature and that the defect is partially expressed at permissive temperatures. During the Percoll density gradient analysis, we found that TfT1.11 cells, in addition to expressing a defect in thetransfer of internalized material to dense lysosomes, also lost P-galactosidase activity from dense lysosomes after incubation at 41 "C (Fig. 2 E ) . This loss was associated with both an increase in the activity found in more buoyant compartments and a decrease in total activity. The vast majority of p-galactosidase activity from WTB cells was observed to sediment in the dense region of 17% Percoll gradients, consistent with localization in late endosomes and dense lysosomes (Fig. 2 F ) . Only a small peak of activity was seen in the buoyant region where early endosomes sediment. For TfT1.ll cells at the nonpermissive temperature, a dramatic increase in activity in the buoyant region was observed, with a corresponding decrease in the dense region. A similar redistribution and loss of activity was observedforP-hexosaminidase (data not shown).Since the time of temperature shift is short relative to the half-lives of lysosomal enzymes,the resultsindicate that mature lysosomal enzymes in TfT1.ll can redistribute from dense lysosomes to compartments with densities corresponding to those of both early and late endosomes. TfTl.11 Cells Fail to Accumulate Dextran at the Restrictive Temperature-The failure of TfT1.ll, following incubation at the nonpermissive temperature, to transfer internalized horseradish peroxidase to dense compartments and to accumulate horseradish peroxidase activity indicated that fluid-phase endocytosis is defective in TfT1.ll. To characterize this defect further, cells were labeled with XRITC-dextran, a fluid-phase marker, for various periods of time and analyzed by flow cytometry for cell-associated XRITC-dextranfluorescence. At 33 "C, both WTB and TfT1.11 accumulated XRITC-dextran at

and TfT1.ll cells regardless of whether they had been preincubated a t 41 "C for 4 h, although a slight increase in buoyant density of transferrin-containing compartments was observed in TfT1.ll after incubation at the nonpermissive temperature. These results indicate that the decreased rate of externalization of transferrin in TfT1.ll at the restrictive temperature does not result from mislocalization of transferrin into late endosomes and dense lysosomes. Incubation at the Restrictive Temperature Results ina Redistribution of p-Galactosidase and Inhibition of Dansferof Znternalized Horseradish Peroxidase to Dense Lysosomes-Since many treatments that inhibit or alter receptor recycling also inhibit or alter the transfer of internalized material to dense we considered the possibility that this corlysosomes (14, 151, relation might hold for TfTl.11. We therefore examined, for TfT1.ll and WTB at the permissive and nonpermissive temperatures, the distribution on both types of Percoll gradients of compartments containing an internalized fluid-phase marker, horseradish peroxidase (Fig. 2). For WTB, endocytosed horseradish peroxidase was found primarily in dense lysosomes at both temperatures. While the total horseradish peroxidase activity present in the gradient increased for WTB at the higher temperature, the fraction of activity in the dense region remained the same. For TfT1.ll at the permissive temperature, endocytosed horseradish peroxidase was also found in dense compartments, although the fraction was somewhat lower than for WTB (Fig. 2A). However, at the nonpermissive temperature, horseradish peroxidase activity was completely absent from the dense lysosome region of 27% Percoll gradients in TfT1.ll (Fig. 2 B ) . The defect appears to be in transferofhorseradish peroxidase out of early endosomes (to late endosomes), since horseradish peroxidase activity was also absent from the dense region of a 17% Percoll gradient (Fig. 2C). These results indicate that TfT1.11 exhibits a defect in the transferof inter-

27% Percoll 41'C

17% Percoll

33'c

41'C

2.5

2.5

2.0

2.0

1.5

1.5

1.o

1.0

0.5

1.5

0.0

1.0

200

m

150

150

100

I00

50

10

0 0

5

10

IS

20

25

300

5

10

15

20

'25

300

5

10

15

20

2S

30

0

Fnctlon number FIG.2. Inhibition of transfer of fluid-phase material to dense lysosomesand redistributionof a lysosomal enzyme to more buoyant compartments in TfTl.ll at 41 "C.WTB (circles)and TfTl.11 (triangles) cells were either maintained at 33 "C (A, D ) or incubated at 41 "C( B , C , E , F)for 4 h and then labeled for 1.5 h with 2.5 mg/ml horseradish peroxidase. Postnuclear supernatants were prepared and fractionated on 27% (A, B , D , E ) or 17% Percoll (C, F ) gradients. Fractions were collected from the top and assayed for horseradish peroxidase ( A X ) and @-galactosidase(D-F). Enzyme activities were normalized to the amount of protein in the postnuclear supernatants.

25360

Receptor Recycling and Lysosome Biogenesis

120 I-

0

1 0 2 0 3 0 4 0 5 0 6 0 0

1 0 2 0 3 0 4 0 5 0 6 0

Labeling time (min)

FIG.3. TfTI.ll cells are temperature conditional for the accumulation of material internalized by fluid-phase endocytosis. WTB (0) and TfT1.ll (V) cells were either maintained at 33 "C (A) or 0 1 2 3 4 5 6 incubated at 41 "C for 4 h( B ) .The cells werethen incubated for various times with 2 mg/ml XRITC-dextran prior to the analysis of cell-associTime at 41'C prior to pulse ated XRITC fluorescence. Data shown are the averages and standard deviations for triplicate samples from two experiments normalized to FIG.4. Kinetics of expression of the decrease in accumulation the WTB average value at 40 min in each experiment. of XFUTC-dextran. WTB (circles) and TfT1.ll (triangles) cells were either maintained at 33 "C (open symbols) orincubated at 41 "C (closed a similar rate and to a similar extent (Fig. 3) (at later times, symbols) for various times and then incubated with 2 m g / d XRITCTfT1.ll accumulated slightly less XRITC-dextran). At 41 "C, dextran for 30 min (at the same temperature as the preincubation). WTB and TfT1.ll initiallyaccumulated XRITC-dextran to the Data shown are the averages and standard deviations for triplicate samples from two experiments normalized to the WTB 33 "C average same extent, but at later timesaccumulation of XRITC-dextran value in each experiment.

by TfT1.ll was greatly diminished and reached saturation after 40 min. The similar initial internalization rate combined with the decreased extent of accumulation suggest that the defect in TfT1.ll is not in internalization, but rather that at the nonpermissive temperature almost all internalized XRITCdextran is rapidly lost from the cells. To determine the kinetics of expression of this defect, cells were shifted to the restrictive temperature for various times, incubated with XRITC-dextran for 30 min, and then analyzed (Fig. 4). Consistent with the results for horseradish peroxidase (Fig. 2),WTB has an increased accumulation of the fluid-phase while the ability of marker at therestrictivetemperature TfT 1.11to accumulate XRITC-dextran during a 30-min pulseis reduced to 35%of WTB at that temperature. Thefull expression of the defect was observed after 2 h at the nonpermissive temperature. TfTl.11 Cells Exhibit a Rapid Eflux of Internalized Dextran a t the Restrictive Temperature-The difference in accumulation of XRITC-dextran between WTB and TfT1.11 is presumably due toa difference in the rate of dextran efflux since the initial extent of accumulation of XRITC-dextran is very similar between the two cell lines. To demonstrate that TfT1.11 has an enhanced efflux of XRITC-dextran at the restrictive temperature, cells were incubated for 4 h at 41 "C, and then for either 5 or 60 min with XRITC-dextran. The cells were washed, incubated in the absence of XRITC-dextran for up to 90 min, and then analyzed for the amount of XRITC-dextran fluorescence retained (Fig. 5). WTB cells labeled for 5 min displayed a n initial rapid loss of 60%of the cell-associated fluorescence followed by a much slower efflux; WTB cells labeled for 60 min displayed a slow loss of cell-associated fluorescence and retained greater than 80% of the XRITC-dextran at 90 min. These results are consistent with the concept that in normal cells fluid is rapidly lost from early endosomes but only slowly lost from later endocytic compartments. In contrast, TfT1.ll labeled for 5 min displayed a n increased loss (80%)of the initial cell-associated fluorescence during the first 10 min of incubation without dextran andlost essentially allfluorescence by 90 min. Also in contrast toWTB, TfT1.11 cells labeled for 60 min lostapproximately 40%of the internalized XRITC-dextran

-

OI.."'.."'...''....'...' 0 20 40 60

'

8 0 0

.

" *

20

'

.40'

"

*

' ..

60

80

.

'

Time after labeling (min)

FIG.5 . TfT1.ll cells fail to accumulate fluid-phase material due to rapid efflux. WTB (circles) and TfT1.11 (triangles) cells were incubated at 41 "C for 4 h, incubated with 2 m g / d XRITC-dextran for 5 (A) or 60 ( B )min, chilled to 0 "C, washed, and then warmed to 41 "C without XRITC-dextran. At the indicated times, the cells were chilled, washed, and analyzed byflow cytometry for XRITC-dextran fluorescence. Data shown are the averages and standard deviations for triplicate samples from twoexperiments and are expressed as the percentage of the initial pulse.

within the first 20 min, and by 90 min had lost greater than 60% of the initial cell-associated fluorescence. These results demonstrate that TfT1.ll has an enhanced efflux of fluidphase material after incubation at the nonpermissive temperato the slowed rate of externalture, and are in sharp contrast ization of transferrininTfT1.llthat we have previously reported (13). Incubation a t the Restrictive nmperature Results in an Enhanced Eflm of Previously Endocytosed Dextran-&r incubation of TfT1.ll at 41 "C, lysosomal enzyme activities were redistributed to more buoyant compartments (Fig. 2). A consequence of this redistribution maybe that material present in dense lysosomes would also redistribute to buoyant compartments and,therefore, mayhave a n increased rate of exocytosis.

Receptor Recycling anId Lysosome Biogenesis

25361

cycling vesicles and late endosomes, thus requiring only one type of fission event. This model has received strong support from complementary experimental approaches (24, 25). Current data strongly suggest that receptor recycling and the delivery of endocytosed material to dense lysosomes are intimately linked. For example, incubation in the presence of weak bases or ionophores (treatments that increase intravesicular pH) inhibit receptor recycling (26-32) and inhibitdelivery of material to dense lysosomal compartments (20, 33, 34). In addition, incubations with weak bases or ionophores also DISCUSSION inhibit fluid-phase pinocytosis (281, and enhance thesecretion of lysosomal enzymes (26). Vanadate has also been shown to Receptor Recycling and lFafic to Lysosomes Are Linked-A central concept in many currentviews of endocytic membrane block receptor recycling and transport to dense lysosomes (35). traffic is a distinction between a t least two classes of acidic Similarly, temperatures below 22 "C inhibit receptor recycling (32,36-38), ligand dissociation (391, and delivery of material to organelles, endosomes and lysosomes. Endosomeswereinitially proposed to be as acidic as lysosomes but lacking in hy- dense lysosomal compartments (34, 37, 40, 41). Although sevdrolases, and exposure of endocytosed material to thesehydro- eral of the low temperature blocks in endocytosis could result lases was thought to occur via fusion with lysosomes after from effects on microtubules, disruption of microtubule funcrecycling of receptors to thecell surface (22).The appealof this tion cannot account for all of the effects of low temperatures elegant andsimple notion was eventuallyovercome by evidence since agents that disruptmicrotubules do not inhibitrecycling that endosomes and lysosomes can differ significantly in pH of transferrin (42, 43) or viral membrane proteins (44). These and that endosomes contain significant hydrolase activity (for agents do inhibit receptor-ligand segregation (39) anddelivery processes can be inhibreview, see Ref. 5). These observations led to the proposal of to lysosomes (45), indicating that these new models to explain how endocytosed material is transported ited without affecting recycling. The converse does not appear from early endosomes to dense lysosomes (reviewed in Refs. 3 to be true, however, since inhibition of recycling is associated and 23). The stable compartmentmodel postulates that mate- with a n inhibition of lysosomal delivery. The results presented here support this conclusion without rial is delivered to pre-existing structures by fusion with priis recycled to thecell the useof drugs or temperatures that disrupt normal cell funcmary endocytic vesicles and that material surface and delivered to late endosomes without theconsump- tions. Incubation of TfT1.11 cells at 41 "C (a temperature at tion of the original organelle. Thus, the stable compartment which the viability and endocytic apparatus of parental WTB model requires two separate fission events to generate thetwo cells are normal) results ina reduction in the rate of receptor different populations of shuttle vesicles while maintaining the recycling and aninhibition of transfer of endocytosed material original structure. The transient compartment or maturation to late endosomes and dense lysosomes. The simplest explanamodel, on the other hand, postulates that endosomes are gen- tion (model 1)is that TfT1.11cells are defective in a component erated by the coalescence of primary endocytic vesicles (and required for proper removal of recycling material (a fission vesicles carrying hydrolases) andconsumed by fission into re- process) and that this removal is requiredfor the generationof a late endosome (which is required for appearance of endocytosed material in lysosomes). This correlation is embodied in the transient compartment model. It is difficult to reconcile with stable compartment models, which postulate two distinct types of fission processes. Therefore, the inhibition of receptor recycling and the loss of transport of material to dense lysosomes in TfT1.ll upon incubation at the nonpermissive temperature provide further evidence that post-sorting compartments andrecycling vesicles are generatedby at least a coupled mechanism if not the samemechanism. Other mutants with defects in lysosome biogenesis have been described previously. The CHO cell mutant, V.24.1, in the End4 complementation group is defective in secretion and lysosome biogenesis but can recycle transferrin normally (46); TfT1.11 and V.24.1 are indifferent complementation group^.^ ADictyostelium discoideum mutant, HMW570, with a defect in fluidphase endocytosis also fails to deliver lysosomal enzyme activitytodense lysosomes (47);whether endosomal recycling occurs in the mutant is unknown. While the relationships between the genes affected in these mutants are not clear, yet the 0 2 4 6 8 10 results suggest that defects in either thesecretory or receptor Time after start of labeling (h) recycling pathway can affect the process of lysosome biogenFIG.6. WT1.11 cells have enhanced efflux of fluid-phase mate- esis. rial internalized at the permissive temperature after incubaPathways of Receptor Recycling--Two pathways for recycling tion at the nonpermissive temperature. WTB (O,O, 0 )and TfT1.ll ( 0 , V, V) were incubated for 1 h with 2 mg/ml XRITC-dextran and of endocytosed ligands from endosomes to the plasma memwashed. One set of samples was analyzed (0, 0 )and the remaining brane have been described. The kinetics of recycling of transsamples were incubated without XRITC-dextran for1 h. The cells were ferrin (13, 48) and epidermalgrowth factor (49)are consistent then either maintained a t 33 "C (0,V) or incubated a t 41 "C (0,V) for with a fast process from early endosomes to the surface (tllz< various times prior to the analysis of XRITC fluorescence. Data shown 10 min) anda slower process that may involve transit through are the averages and standard deviations for triplicate samples from

To test this hypothesis, cells were labeled at 33 "C for 1h with

XRITC-dextran and then incubated in theabsence of XRITCdextran for 1 h prior to incubation at 41 "C for up to 6 h. TfT1.11 lost about 70% of the initial cell-associated XRITCdextran by 6 h after temperature shift (Fig. 6), while WTB at 41 "C and both TfT1.ll and WTB maintained at 33 "C, on the other hand,lost only 40-50% of the initialfluorescence by 6 h. Thus, TfT1.ll exhibits enhancedexocytosis of material internalized prior to incubation at the elevated temperature.

two experiments. Values are expressed as a percentage of the average WTB value with no chase.

a R.

B. Wilson and R. F. Murphy, unpublished results.

25362

Receptor Recycling and Lysosome Biogenesis

the Golgi apparatus (apparent overall t y 2 > 60 min). In the cycled for deliveryto early endocytic compartments (5,56). The second process,the lateendosome mayserve as a salvage com- results presented here are consistent with the idea that lysopartment to return missorted receptors back to the cell surface somal enzymes may normally be recycled back to earlier endoby way of the Golgi apparatus (50). cytic compartments and that as a result of the temperatureRecent results indicate that the receptor-recycling pathway conditional defect in the transportof material from endosomes from the endosome backto the cell surface is a default pathway to later endocytic compartments, recycling lysosomalenzymes (10,51).The default pathway, or bulk flow, model predicts that are trapped in buoyant compartments. It would be expected recycling receptors should travel from the endosome backto the that endocytosed material present within lysosomes would recell surface at similar rates, as has been observed for several cycle along with the enzymes to earlier compartments and that receptors (52). In thismodel, integral membrane proteins, such there would be an increase in the exocytosis of previously inas the epidermal growth factor receptor, the IgG Fc domain ternalized material. Therefore, the enhanced efflux of previreceptor, and the mannose 6-phosphate receptor, that are re- ously endocytosedmaterial andthe redistribution of lysosomal tained in theendosome and camed further in pathway the (24, enzymes observed in TfT1.ll at the nonpermissive tempera32, 53, 54), would be predicted to require specific signals for ture suggests that dense o r late endocytic compartments are in retention within the vacuolar component of the endosome. It communication by a retrograde pathway with earlier, more would also seem likely that a signal would be required to iden- buoyant compartments. tify the post-sorting vacuole forfurther processing in theendoConclusion-The analysis of TfT1.11 presented here shows a cytic pathway. Recently, Goltz et al. (55) have successfully re- link between receptor recycling and lysosome biogenesis and constituted the fission of endosomes into post-sorting vacuoles suggests that mature lysosomal enzymes may recycle to endoand recycling tubuledvesicles. Their results indicate that the somes. We have presented two possible explanations of the vacuolar component of the endosome is specifically identified TfT1.ll phenotypes, inhibition of endosomal fission or misloand that the binding of cytoplasmic dynein to the endosomal calization of a retention signal. Future experimentation will be membrane may be involved in this identification. The results required to determine which, if either, of these models is coralso suggest that the fission of the endosome into recycling rect. In particular, identification of the protein or proteins that tubuledvesicles and post-sorting vacuoles maybe mediated by are altered in TfT1.ll (either by biochemical or molecular biothe interaction of dynein bound to endocytic compartments and logical methods) should be of significant value for understandmicrotubules. ing the mechanisms by which the crucial processes of receptor Receptor recycling is slowed in TfT1.ll after incubation at recycling and lysosome biogenesisare accomplished. the nonpermissive temperature (13),while release of an interAcknowledgments-We thank Dr. Rockford K. Draper for helpful disnalized fluid-phase marker is enhanced (Fig. 5). To explain this apparent contradiction, we hypothesize that, if fission is defec- cussions and Greg LaRocca for technical assistance. tive in TfT1.ll as proposed in model 1, then there exists a REFERENCES mechanism for removalof fluid from endosomes following seg- 1. Hubbard, A. L. (1989) Cur,: Opin. Cell Biol. 1, 675-683 regation of receptors into recycling tubules. An alternative hy2. Rodman, J. S.,Mercer, R. W., and Stahl, P. D. (1990) Cur,: Opin. Cell Biol. 2, 664-672 pothesis (model 2) would be that inTfT1.ll at thenonpermis3. Murphy, R. F. (1991) Der& Cell Biol. 1, 77-82 sive temperaturethe signal that identifies the vacuolar 4. Mellman, I., Fuchs. R., and Helenius, A. (1986) Annu. Reu. Biochem. 65, 663-700 component of the endosome as a compartment for retention in 5. Murphy, R. F. (1988) Adu. Cell Biol. 2, 159-180 the pathway to lysosomes is allowed to enter the tubular exR. F. (1989)Proc. Natl. Acad. Sci. U.S. A. 6. Cain, C. C., Sipe, D. M., and Murphy, tensions of the endosome. After fissionof the tubules from the 86,544548 7. Fuchs, R., Sehmid, S., andMellman, I. (1989)fioc. Natl. Acad. Sci. U. S. A. 86, vacuolar component, the mislocalized signal would divert the 53s543 tubular extensions, which contain receptors, from the constitu8. Geuze, H. J., Stoorvogel, W.,Strous, G . J., Slot, J. W., Eleekemolen. J. E.,and Mellman, I. (1988) J. Cell Biol. 107,2491-2501 tive recycling pathway to the lysosome-directed pathway. At 9. Linderman, J. L., and Ladenberger, D. L.(1988) J. Theo,: Biol. 182,203-245 this stage, receptors might then be recycled back to the cell 10. Dunn, K. W., Maraw, T. E., andMaxfield,F. R. (1989) J. Cell B i d . 109, surface byway of the salvage pathway for the recycling of 3303-3314 receptors through the Golgi apparatus (50). This is consistent 11.Colbaugh, P. A,, andDraper, R. K. (1993) Adv. Cell Mol. Biol. Memb,: 1, 169-198 with the observation that only the fast receptor recycling proc- 12. Whitters. E.A,. Skinner., H. B... and Bankaitis, V A. (1993)Mu. Cell Mol. Biol. Memb,: I, 367-339 ess is inhibited in TfT1.ll (13). The defect would reduce the C. C . , Wilson, R. B., and Murphy, R. E (1991) J. Bioi. Chem. 266, rate of externalization of receptors, and thus reduce surface 13. Cain, 1174611752 receptor number as seen in TfT1.11at elevated temperatures. 14. Berg, T., Blomhoff,R., Naess, L., lblleshaug, H., and Drevon, C.A. (1983)Exp. Cell Res. 148, 319-330 Since the vacuolar component or post-sorting endosome would 15. Chang, P. L., Amen, M., Yu, C. Z., and Kelly. E. M. (1988) Exp. Cell Res. 176, no longer contain specific retention signals, it may follow the 258-267 default pathway and be delivered to the plasma membrane. 16. Roederer, M., Mays, R. W., and Murphy,R. F. (1989)Eur. J . Cell Biol. 4 8 , 3 7 4 4 17. Markwell, M. A. K. (1982) A w l . Biochem. 126,427432 The overall effect of this defect would beto increase the rateof 18. Robbins, A. R. (1979) Proc. Natl. Acad. Sci. U. S. A. 76, 1911-1915 effluxof material internalized by fluid-phase endocytosis which 19. Steinman, R. M., and Cohn, Z. A. (1972) J . Cell Biol. 55, 18G204 is normally retained in the vacuolar component of the endo- 20. Merion, M., and Sly, W. S.(1983) J. Cell Biol. 96.644-650 21. R o f f , C. F., Fuchs, R., Mellman, I., and Robbins, A. R. (1986) J. Cell Biol. 103, some. It is interesting to note that a D.discoideum mutant that 2283-2297 e & Biochem. is defective in fluid-phase endocytosis also fails to deliver lyso- 22. Helenius, A,, Mellman, I., Wall, D., and Hubbard, A. (1983) D Sci. 8, 245-249 somal enzyme activity to dense lysosomes (47). 23. Griffiths, G., and Gruenberg, J. (1991) Der& Cell Biol. 1, &9 Recycling of Mature Lysosomal Enzymes to Endosomes-In 24. Stoorvogel, W., Geuze, H. J., Griffith, J. M., Schwartz,A. L., and Strous, G . J. (1989) J . Cell B i d . 108,2137-2148 addition to the defect in transport of material to dense lyso25. Dunn, K W., and Maxfield, F. R. (1992) J. Cell Biol. 117,301-310 somes foundin TfTl.11 after incubation at the nonpermissive 26. Gonzalez-Noriega, A,, Grubb, J. H., Talkad, V., and Sly, W. S. (1980) J. Cell Biol. 85, 63-52 temperature, we observed that lysosomal enzyme activities S. K., Goldstein, J. L., Anderson, R. G. W.,and Brown, M. S. (1981) Cell were redistributed to more buoyant compartments and mate- 27. Basu, 24,493-502 rial internalized prior t o a temperature shift was selectively 28. Kaplan, J., and Keogh, E.A. (1981) Cell 24,925-932 lost. Previous studies have indicated that in addition to the 29. Ciechanover,A.. Schwartz,A. L., Dautry-Varsat,A,, and Lodish,H. F. (1983) J. Biol. Chem. 258,9681-9689, deliverv of newly svnthesized lysosomal enzymes to early en- 30. Strous, G . J., Maine, A. D., Zgderhand-Eleekemolen. J. E., slot, J. w., and Shwnrtz. docytic compartments, older lysosomal enzymes may be re.. - ", A. L. (1985) J. Cell Bioi. 101.531-539 ~~~

"

~~~~~~

Recycling Receptor

and Biogenesis Lysosome

25363

31. Stoorvogel, W., Geuze, H. J., and Strous, G. J.(1987)J. Cell Biol. 104, 1261- 43. Sakai, T.,Yamashina, S., and Ohnishi, S. (1991)J. Biochem. (Tokyo) 109, 1268 52&533 32. Felder, S., Miller, K.. Moehren, G., Ullrich, A,, Schlessinger, J., and Hopkini4. Gruenberg, J., Griffiths, G., and Howell, K. E. (1989)J. Cell Biol. 108,13011316 C. R. (1990)Cell 61,623434 33. Harford, J.,Wolkoff, A. W., Ashwell, G., and Klausner,R. D. (19831J. Cell Biol. 45. Oka, J. A,, and Weigel, P. H. (1983)J. B i d . Chem. 258, 10253-10262 96, 1824-1828 46. Colbaugh, P. A., Stookey, M., andDraper, R. K. (1989)J. Cell Biol. 108, 34. Sullivan, P. C., Ferris, A. L., and Storrie,B. (1987)J. Cell. Physiol. 131,5843 2211-2219 35. Kindberg, G. M., Gudmundsen, O., and Berg, T.(1990)J . Biol. Chem. 265, 47. Ebert, D. L., Freeze, H. H., Richardson, J., Dimond, R. L., and Cardelli, J. A. 8999-9005 (1989)J . Cell B i d . 109, 1445-1456 36. Sipe, D. M., and Murphy, R.F. (1987)Proc. Natl. Acad. Sci. U. S . A . 84, 48. Stein, B. S., and Sussrnan, H. H. (1986)J . Biol. Chem. 261, 10319-10331 7119-7123 49. Sorkin, A., Krolenko, S., Kudrjavtceva, N., Lazebnik, J., Teslenko, L., Soder37. Harding, C. V.,and Unanue, E.R. (1990)Eur J. Irnmunol. 20, 323-329 quist, A. M., and Nikolsky, N. (1991)J. Cell B i d . 112, 5543 38. Sipe, D. M., Jesurum, A., and Murphy, R. F. (1991)J . B i d . Chem. 266, 346% 50. Green, S. A,, and Kelly, R. B. (1992)J . Cell Biol. 117, 47-55 3474 51. Mayor, S., Presley, J. F., and Maxfield, F. R. (1993)J . Cell Biol. 6, 1257-1269 39. Wolkoff, A. W., Klausner, R. D., Ashwell, G.,and Harford, J. 11984)J. Cell Biol. 52. Ward, D. M., Ajioka, R., and Kaplan, J. (1989)J.Biol. Chern. 264, 8164-8170 98,375481 53. Mellrnan, I., and Plutner, H. (1984)J . Cell Biol. 38, 1170-1177 40. Dunn, W.A., Hubbard,A. L., andAronson, N. N., Jr. (198O)J.Biol. Chem. 256, 54. Honegger, A. M., Dull, T.J., Felder, S., Van Obberghen, E., Bellot, F., Szapary, 5971-5978 D., Schmidt, A,, Ullrich, A,, and Schlessinger,J. (1987)Cell 51, 199-209 41. Hopkins, C. R., and Trowbridge, I. S. (1983)J . Cell Biol. 97, 50%521 55. Goltz, J.S., Wolkoff, A. W., Novikoff, P. M., Stockert, R. J., and Satir,P. (1992) 42. Hedley, D.W., and Musgrove, E. A. (1986)Biochem. Biophys. Res. Commun. Proc. Natl. Acad. Sci. U. S. A. 89, 70267030 138, 12161222 56. Bowser, R.,and Murphy, R. F. (1990)J. Cell. Physiol. 143, 110-117