Cell, Vol. 70, 887-900,

September

18, 1992, Copyright

0 1992 by Cell Press

CLIP-1 70 Links Endocytic to Microtubules

Vesicles

Philippe Pierre, l Jochen Scheel, l Janet E. Rickard,’ and Thomas E. Kreis’ European Molecular Biology Laboratory Meyerhofstrasse 1 D-6900 Heidelberg Germany

Summary Binding of endocytic carrier vesicles to microtubules depends on the microtubule-binding protein CLIP-170 in vitro. In vivo, CLIP-170 colocalizes with a subset of transferrin receptor-positive endocytic structures and, more extensively, with endosomal tubules induced by brefeldin A. The structure of CLIP-170 has been analyzed by cloning its cDNA. The predicted nonhelical C- and N-terminal domains of the homodimeric protein are connected by a long coiled-coil domain. We have identified a novel motif present in a tandem repeat in the N-terminal domain of CLIP-170 that is involved in binding to microtubules. This motif is also found in the Drosophila Glued and yeast BlKl proteins. These features, together with its very elongated dructure, suggest that CLIP-1 70 belongs to a novel class of proteins, cytoplasmic linker proteins (CLIPS), mediating interactions of organelles with microtubules. Introduction Extracellular material and plasma membrane proteins are endocytosed at the cell surface via coated pits and transferred to early endosomes, where they are sorted either for delivery to late endosomes and lysosomes or for recycling to the plasma membrane (for reviews see Rodman et al., 1990; Gruenberg and Howell, 1989; Kornfeld and Mellman, 1989). The microtubule network, which plays important roles in intracellular membrane traffic and the spatial organization of cytoplasmic organelles, is involved in the endocytic pathway. Transport from early to late endosomes depends on microtubules (Gruenberg et al., 1989; De Brabander et al., 1988; Matteoni and Kreis, 1987; Herman and Albertini, 1984), while internalization of material into early endosomes and its recycling to the plasma membrane are not affected by treatment of cells with nocodazole (Gruenberg et al., 1989). Depolymerization of microtubules by nocodazole leads to accumulation of internalized material in discrete endocytic vesicles that are the putative carriers between early and late endosomes (Gruenberg et al., 1989). Microtubules are also essential for clustering of late endosomes and lysosomes in the pericentriolar region, a position that ensures optimal intercompartmental transfer of material (Scheel et al., 1990). Furthermore, microtubules are required for the formation

* Present address: Department of Cell Biology; Ill; 30, quai Ernest-Ansermet; CH-1211 Geneva

University, Sciences 4; Switzerland.

of tubular extensions from endosomes and lysosomes in cells treated with brefeldin A (BFA; Lippincott-Schwartz et al., 1991; Wood et al., 1991; Hunziker et al., 1991). Proteins mediating interactions between cellular organelles and microtubules have been identified in various Cell-free assays, and several approaches are being used to elucidate the molecular mechanisms underlying the interaction of endosomes with microtubules. The microtubule-based motor proteins kinesin and cytoplasmic dynein, which mediate organelle movement along microtubules (for reviews see Vale, 1992; Schroer and Sheetz, 1991; Vallee and Shpetner, 1990), are involved in the microtubule-dependent fusion of apical and basolateral endosomes isolated from polarized Madin-Darby canine kidney cells (Bomsel et al., 1990). Kinesin is also involved in the formation of tubular extensions from lysosomes (Hollenbeck and Swanson, 1991). Dynamin, another microtubule-based motor protein (Shpetner and Vallee, 1989) may play a role in endocytosis, since it is homologous to the Drosophila protein shibire (Chen et al., 1991; van der Bliek and Meyerowitz, 1991), mutants of which are affected at an early step in endocytosis (Kosaka and Ikeda, 1983). The perinuclear localization of lysosomes in thyroid cells may involve a 50 kd protein mediating binding of lysosomes to microtubules in vitro (Mithieux and Rousset, 1989). It has also been shown that endocytic carrier vesicles that accumulate in cells treated with nocodazole bind to microtubules in vitro; this binding is cytosol dependent and nucleotide sensitive, but independent of kinesin and cytoplasmic dynein (Scheel and Kreis, 1991). A role for microtubule-binding proteins (in addition to the motor proteins) has therefore been suggested (Scheel and Kreis, 1991; see also Schroer and Sheetz, 1991). These postulated microtubule linker proteins could be involved in the capture of early endosomes prior to their inward translocation by motor proteins, and in addition they may regulate the activity of these motor proteins. Here we show that a 170 kd microtubule-binding protein from HeLa cells (Rickard and Kreis, 1990) is essential for the binding of endocytic vesicles to microtubules in vitro and that this protein colocalizes with endocytic organelles in vivo. Cloning and sequencing of the cDNA for this protein show that it is a novel protein. Mutational analysis has identified a novel motif involved in microtubule binding; this motif appears to be highly conserved, since it is also found in rat DP-150 (Holzbaur et al., 1991), the Drosophila Glued protein (Swaroop et al., 1987), and BlKl, a yeast microtubule-binding protein (Berlin et al., 1990). Because of the role of this protein in mediating endocytic vesiclemicrotubule interactions and its apparent molecular size, we call it cytoplasmic linker protein, CLIP-170. Results CLIP-170 Is Involved in the Binding of Endocytic Carrier Vesicles to Microtubules In Vitro Binding of horseradish peroxidase (HRP)-labeled endocytic carrier vesicles to microtubules depends on organ-

elle membrane proteins, is nucleotide sensitive, and is mediated by cytosolic microtubule-binding proteins different from the microtubule-based motors kinesin and cytoplasmic dynein (Scheel and Kreis, 1991). To analyze the involvement in this binding of a 170 kd nucleotide-sensitive microtubule-binding protein (Rickard and Kreis, 1990, 1991) here designated CLIP-170, HeLa cytosol was immunodepleted of this protein using two murine monoclonal antibodies (MAbs), 3A3 and 2D6, recognizing different epitopes (J. E. Ft. and T. E. K., unpublished data). Preincubation of HeLa cytosol with either of these MAbs coupled to Sepharose beads depletes the cytosol of CLIP-170 as judged by immunoblotting with a third MAb, 4D3, whereas incubation of cytosol with beads carrying a control MAb directed against kinesin (SUK4; lngold et al., 1988) does not affect the concentration of CLIP-170 (Figure 1A). HeLa cytosol immunodepleted of CLIP-170 no longer supports endocytic carrier vesicle binding to microtubules, whereas cytosol pretreated with SUK4 is still fully active (Figure 16). The amount of CLIP-170 in the cytosol correlates with its activity in the binding assay during fractionation of HeLa cytosol with ammonium sulfate. Precipitation of cytosolic protein with 40% ammonium sulfate leaves 30% of CLIP170 in the supernatant. Correspondingly, the binding activity of this supernatant is reduced to about one third of the value of control cytosol (Figure 1). The specific immunodepletion of CLIP-170 from cytosol and ammonium sulfate fractionation suggests that CLIP-170 is involved in the binding of endocytic carrier vesicles to microtubules. CLIP-170 was immunopurified from HeLa cytosol using the MAb 3A3 (Rickard and Kreis, 1991) to confirm its involvement in the binding of endocytic carrier vesicles to microtubules. Addition of affinity-purified CLIP-170 to cytosol depleted of CLIP-1 70 (using MAb 3A3) restores the ability to support binding in the assay in proportion to the concentration of added protein (Figure 1 A, lanes 1 and 4-6). Binding could not be fully restored, most likely because only an insufficient concentration of CLIP-1 70 could be achieved to replenish its normal cytoplasmic concentration. The same results were obtained when cytosol was depleted of CLIP-1 70 using MAb 2D6. Rescue of activity of CLIP-l 70-depleted cytosol is also abolished upon heat treatment of CLIP-170. Purified CLIP-170 alone is unable to promote binding of endocytic carrier vesicles to microtubules (data not shown), indicating the involvement of additional cytosolic factors. Furthermore, binding of another class of vesicles, derived from the transQolgi network, to microtubules in vitro (van der Sluijs et al., 1990) is independent of CLIP-170 (D. Hennig and T. E. K., unpublished data). Taken together, these data strongly suggest a specific role for CLIP-1 70 in the interaction of endocytic carrier vesicles with microtubules. CLIP-170 in HeLa

Colocalizes Cells

with

Endosomes

We have previously reported that patches of CLIP-170 accumulate at the peripheral ends of a subset of microtubules (Rickard and Kreis, 1990). Further analysis of the distribution of CLIP-170 in HeLacells by confocal immunofluorescence microscopy and three-dimensional image re-

1

2

cytosol

-

+

depletion

-

CLIP-170

-

Figure 1. CLIP-1 70 Mediates cles to Microtubules In Vitro

3

4

5

6

7

+

+

+

+

+

3A3

2D6

SUK4

3A3

AS

-

-

-

+

-

the Binding

of Endocytic

Carrier

Vest-

HeLa cytosol was preincubated with Sepharose beads coupled with MAb against CLIP-170 (3A3, 2D6) or a control MAb (SlJK4). Alternatively, cytosolic proteinswere precipitated with 40% ammonium sulfate (AS). CLIP-170 was affinity purified from HeLa cytosol as described in Experimental Procedures. (A) The amount of CLIP-170 in untreated cytosol (lane 2) in immunodepleted cytosols without further additions (lane 3-5) or after addition of purified CLIP-170 (lane 6) and in the 40% ammonium sulfate supernatant (lane 7) was estimated by immunoblotting with MAb 4D3. (8) The binding to microtubules of saltwashed endocytic carrier vesicles previously loaded with HRP was measured in the absence of cytosol (lane 1) and in the presence of 2 mglml untreated (lane 2) or an equivalent volume of pretreated HeLa cytosol (lane 3-7). Binding was determined by measuring HRP activity in bound and unbound fractions.

construction reveals additional patchy structures distributed throughout the cytoplasm (Figures 2a and 2a’). Since CLIP-170 is involved in the interaction of endocytic vesicles with microtubules in vitro, we investigated whether CLIP-1 70 colocalizes with endosomal organelles in HeLa cells. We have so far been unsuccessful in using our antibodies against CLIP-170 for immunoelectron microscopy, and since no specific markers for visualizing endocytic carrier vesicles by light microscopy are available, we have used transferrin receptor (TFR) as an immunofluorescence marker for endosomes in control and BFA-treated HeLa cells. TFR is predominantly found in early stages of the endocytic pathway, in coated pits and early endosomes (Hopkins and Trowbridge, 1983) that presumably do not interact with microtubules (Gruenberg et al., 1989). As shown in Figures 2b and 2c, only a small proportion of CLIP-170 colocalizes with TFR in control HeLa cells, However, more extensive interaction of TFR-positive endosomes with microtubules is observed in cells treated with BFA, which induces the microtubule-dependent formation of TFR-containing membrane tubules (LippincottSchwartz et al., 1991; Wood et al., 1991; Hunziker et al., 1991). A significant colocalization of CLIP-170 and TFR can be observed in the BFA-treated cells, although a frac-

t;;P-170

Links

Endosomes

to Microtubules

Figure 2. lmmunofluorescence Localization of CLIP-170 and Transferrin Receptor in HeLa Cells HeLa cells were fixed with methanol, and CLIP-170 (a, a’, b, and d) and TFR (c and e) were visualized by immunofluorescence microscopy. (a and a’) Stereo reconstruction from optical sections recorded in the confocal microscope at 0.4 urn intervals of HeLa cells labeled for CLIP-170. Note the patchy labeling throughout the cytoplasm in addition to the stronger labeling at the periphery, probably at ends of microtubules. The stereo images should be viewed with stereo glasses. HeLa cells were fixed without pretreament (b and c) or after incubation with 5 rig/ml BFA for 2 min at 37OC (d and e), and CLIP-l 70 and the TFR were localized by double immunofluorescence and visualized by conventional fluorescence microscopy (b and c) or confocal microscopy (d and e). In (d) and (e), single horizontal focal planes near the coverslip are shown. Arrows indicate structures containing both CLIP-l 70 and TFR. Bars, 10 pm.

tion

of each

(Figures

marker

2d

and

is still 2e).

confined

The

low

to distinct

extent

structures

of colocalization

of

and CLIP-170 in untreated cells presumably reflects the predominant localization of TFR in structures of the early stages of the endocytic pathway, which do not interact with microtubules (Gruenberg et al., 1989). In addition, the interaction of endosomes with microtubules mediated by CLIP-170 may be transient, with only a fraction of the total population binding CLIP-170 at any one time. The colocalization of CLIP-170 with endosomal structures competent to interact with microtubules is, however, con-

TFR

sistent

with

the results

of the

in vitro

binding

ing a role for CLIP-170 in the interaction with microtubules in vivo.

assay,

indicat-

of endosomes

cDNA Cloning and Sequence Analysis of CLIP-170 We have cloned the cDNA encoding CLIP-170 to characterize

the

other

proteins

structure

of this

implicated

protein

and

in the

interaction

to compare

it with

of organelles

with microtubules. Five clones were isolated by screening a HeLa cDNA expression library with a mouse polyclonal antiserum raised against ATP-sensitive microtubule-binding proteins from HeLa cells (Rickard and Kreis, 1990). Northern analysis of HeLa poly(A)’ mRNA using the longest of these clones (clone 55) shows a single band at 8

kb (data not shown). Since the five clones had overlapping sequences not covering the entire length of the CLIP-170 open reading frame, clone 55 was used to rescreen the same and two other HeLa cDNA libraries. Forty-five clones were analyzed and classified, and a full-length open reading frame was constructed from restriction fragments of three clones (Figure 3; also see Experimental Procedures). The sequence of the isolated cDNA reveals an open reading frame of 4176 bp encoding a protein of 1392 aa with a calculated molecular weight of 157,000 (Figure 4). The first ATG codon in the open reading frame (position 156158) is close to a second potential initiation codon (position 182-184) with a stop codon 16 bp upstream of the first ATG in the 5’ noncoding sequence (position 141143; asterisks in Figure 4). Neither of the sequences surrounding the two ATG codons conforms well to the consensus sequence for eukaryotic translation initiation (Kozak, 1987) except for the conserved purines at position -3 (positions 153 and 159). Since neither of these codons is a better candidate for initiating translation, we assume that translation starts at the first ATG. Attempts to confirm this by direct sequencing of the N-terminus of CLIP-170 purified from HeLa cells were unsuccessful, presumably because it is blocked. The open reading frame is terminated by tandem stop codons at position 4332-4337 (asterisks

Cdl 890

Figure 3. Restriction Map of the Full-Length CLIP-170 cDNA Clone Schematic representationof cDNAclonesEA2, RDAl, Ml, Hl, and 55. The full-length construct used for the in vitro microtubule binding assays was assembled using clones Ml, 8A2, and RDAl (see Experimental Procedures). Clone Hl, which contains a longer 5’ untranslated region than clone Ml, was used to assemble the sequence shown in Figure 4. Clone 55 was used for production and purification of polyclonal antibodies. The restriction map of CLIP-170 cDNA is represented at the top; the stippled region corresponds to the open reading frame.

Clone 8A2 Clone FCDlA ClmeMl

CloneHI

I

.

.

Clone 55

in Figure 4). No polyadenylation signal could be found in the 3’ noncoding region; the cDNA must not cover the entire length of the CLIP-170 mRNA. Several lines of evidence indicate that the isolatedcDNA encodes CLIP-170. The protein produced from the fulllength cDNA by transcription and translation in vitro comigrates with CLIP-170 from HeLa cells by one- and twodimensional polyacrylamide gel electrophoresis and can be immunoprecipitated using anti-CLIP-170 antibodies (data not shown). The pl of 5.16 calculated from the sequence of CLIP-170 is consistent with the pl of 5.4 determined for dephosphorylated CLIP-1 70 from HeLa cells by two-dimensional gel electrophoresis (Table 1). Microsequencing of tryptic peptides prepared from purified HeLaCLIP-170 identified three fragments that are all present in the predicted sequence (Figure 4, underlined), each of them preceded by a lysine or an arginine, as expected. Antibodies raised against eight peptides derived from the cDNA sequence (Figure 4, dotted lines) recognize CLIP170 by immunoblotting in total HeLa cell extracts as well as in preparations of HeLa microtubule-binding proteins. This positive reaction is abolished by preincubation of the affinity-purified antibodies with the corresponding peptides. The anti-KNDG antibody, raised against a peptide covering amino acids 127-l 41, as well as anti-KMRL (619-638) and anti-NYDS (1315-1330), immunoprecipitate CLIP-170 from HeLa cell lysates (data not shown). Thus, the isolated cDNA encodes CLIP-1 70. The PROSITE data library (release 8.0) was searched for potential posttranslational modification sites in CLIP170 and revealed numerous consensus phosphorylation sites for casein kinase II, protein kinase C, and CAMPdependent protein kinase. CLIP-1 70 is phosphorylated on serine residues in vivo (Rickard and Kreis, 1991) but it is not known at present which serines are phosphorylated or which kinases act on the protein in vivo. Possible phosphoacceptor sites could be the serine residues clustered in the N-terminal part of CLIP-170 (amino acids 38-48,

.Figure

4. Nucleotide

and Deduced

155-175, and 310-340). A leucine zipper pattern is also found between amino acids 1223 and 1244; the presence of this motif may be related to the coiled-coil structure of CLIP-170 described below. No known consensus nucleotide-binding motifs were found, consistent with the lack of a direct effect of ATP on the binding of CLIP-170 to microtubules (Rickard and Kreis, 1991). The predicted secondary structure of CLIP-170 suggests that there are three major regions (Figure 5). An a-helical domain of 960 aa (350-1310) containing heptad repeats (Lupas et al., 1991) is flanked by two non-a-helical domains. The N-terminal domain (amino acids l-349) is basic, with a calculated pl of 10.6, while the a-helical central domain and the C-terminal domain (amino acids 13111392) have acidic pls of 4.8 and 4.2, respectively (Figure 5). Comparison of CLIP-170 with itself using a sensitive

Table

1. Characteristics

of CLIP-170

Sequence data: Polypeptide molecular weight Number of ammo acids Predicted pl Physical measurements: Measured pl Sedimentation coefficient, Diffusion coefficient, D,, Stokes radius (nm)

156,794 1392 5.16 5.3-5.45”

sgoW( x 10%) ( x IO7 cm%)

Calculated properties: Native molecular weight Number of subunits Axial ratio Estimated dimensions (nm) For details of physical measurements mental Procedures. a Value depends on phosphorylation ’ Range is 4.7-6.7. c Range IS 1.56-l .71. d Range is 242,000-376,000.

5.7b

1.64’ 12.2 306,000” 2 45 2.5 by 110

and calculations,

see Experr-

state.

~__~ Amino Acid

Sequence

of CLIP-170

Underlined regions indicate matches of partial protein sequences of HeLa CLIP-170 with the deduced ammo acid sequence. Dotted lines indicate peptides used for raising antibodies; asterisks denote the stop codons. The boxed sequences indicate a repeated motif in the N-terminal domain of CLIP-170. Arrows indicate the beginning and the end of the central a-helical region containmg heptad repeats, and circles indicate the putative metal-binding motif.

CLIP-170 991

Links

Endosomes

to Microtubules

Cell 892

Charge

prorile

(PI) Figure

5. Secondary

1310 I I

350 I I

0 Amino acid number

I

t

10.6

Structure

Predictions

I

1392 I

I 4.8



4.2

I

for CLIP-170

The secondary structure of CLIP-170 was predicted using the method of Gamier et al. (1978). The structure predicted for a region is denoted by the position of the line in the appropriately labeled graph (turns, a helices. or p sheets). The upper line indicates the numbers of amino acid residues; the lower line shows the pi’s of the different domains. CLIP-170 is represented with three domains: the N-terminal domain containing the repeated motif of 57 aa (striped boxes) and the serine clusters (SS), the a-helical region containing the heptad repeats (stippled), and the C-terminal domain containing the potential metal-binding motif (dotted).

homology search algorithm (Argo& 1987) reveals a motif of 57 aa that occurs twice in the basic N-terminal domain (amino acids 57-114 and 210-267). These repeats are 54% identical and contain 20% conservative substitutions (Figure 6a; see also below). Comparison of CLIP-170 with Known Proteins Potential protein sequence similarities between CLIP170 and known proteins were analyzed by searching the GenBank EMBL (release 29) and SwissProt (release 21) data libraries using FASTA and BLAST. No overall homologyof CLIP-170toanyprotein in thedata baseswasfound, indicating that it is a novel protein. Several motifs of CLIP170 were, however, identified in known proteins with a microtubule-related function. These similarities were further investigated by pairwise comparisons of the proteins (see below) with CLIP-170 using sensitive homology search matrices (Argos, 1987). Pairwise comparisons were also performed to search for similarities with known microtubule-interacting proteins. None of these revealed any significant similarity of CLIP-170 with the microtubuleassociated proteins MAPlB (Noble et al., 1989), MAP2 (Lewis et al., 1988), MAP4 (Aizawa et al., 1990; West et al., 1991), tau (Lee et al., 1988) the Drosophila 205 kd MAP (Irminger-Finger et al., 1990), dynein heavy chain (Gibbons et al., 1991) dynamin (Obar et al., 1990), or the microtubule-binding site of trypanosomal MARPs (Schneider et al., 1988). The protein sequence of the tandem repeat in the N-terminal domain of CLIP-1 70 (Figure Sa) is strikingly similar to a single motif in DP-150 (Holzbaur et al., 1991; Figure 6b), Glued (Swaroop et al., 1987), and BlKl (Trueheart et al., 1987; Figure 6~). DP-150 is a rat 150 kd dyneinassociated protein that may be involved in dynein-dependent vesicle movement along microtubules owing to its similarity to dynactin (Gill et al., 1991). The Glued protein, which is 53%~ similar to DP-150 (Holzbaur et al., 1991) is likely to be the homologous protein in Drosophila melanogaster. BIKl, a 60 kd protein from Saccharomyces cerevisiae, is required for microtubule-related functions

during mitosis and colocalizeswith tubulin by immunofluorescence (Berlin et al., 1990). A multiple alignment of the sequences around this motif is shown in Figure 7a. A stretch of 40 aa is highly conserved in BIKl, Glued, DP-150, and both repeats of CLIP-170 (82-l 12 and 226266), showing more than 60% similarity between each of these proteins. Within these 40 aa, 11 aa are absolutely conserved between all these proteins. One of the most prominent conserved motifs is GKN(D/S)G (amino acids 96-100 and 251-255). In addition to these features, CLIP170 has three serine clusters (amino acids 38-48, 155175, and 310-340) at positions equivalent to serine clusters found in the Glued protein (Swaroop et al., 1987). The a-helical domain of CLIP-1 70 shows similarity to a number of filamentous proteins forming coiled-coil structures. The myosin heavy chain A of Caenorhabditis elegans (Dibb et al., 1989) is 52% similar to CLIP-170 over 499 aa in the coiled-coil domain. In addition, tropomyosins, lamins, and proteins of the kinesin superfamily were identified in the data library search (data not shown). The similarity of CLIP-170 to these proteins resides in the coiled-coil domains. Thecentral domainsof DP-150, dynactin, Glued, and BlKl , all predicted to form coiled coils, are also similar to the central domain of CLIP-l 70 as well as to myosin-like proteins (Figures 6b and 6~). The abundance of heptad repeats (Lupas et al., 1991) and the absence of helixbreaking prolines (except one at position 493) in the a-helical domain of CLIP-170 strongly suggest formation of a dimeric coiled coil in this central domain of CLIP-1 70. Further structural analysis will, however, be necessary to corroborate this prediction. The formation of a dimeric structure is also consistent with the shape and oligomerization state of HeLa CLIP-1 70 in solution as deduced from its hydrodynamic properties. The sedimentation coefficient of CLIP-170, determined by sucrose gradient centrifugation, is 5.7 S, and the diffusion coefficient, measured by gel filtration, is 1.64 x 10e7 cm% (Table 1). The same values were obtained for CLIP170 in total HeLa cytosol and for immunopurified HeLa CLIP-l 70, which appears not to be associated with other

CLIP-170 893

Links

Endosomes

to Microtubules

Figure 6. Comparison of CLIP-170 with Itself, DP-150, and BlKl Using Homology Search Matrices The window lengths of the homology search matrices ranged from 5 to 35 in steps of 2 with a stringency of 40. (a) Comparison ofCLIP70 with itself, showing the repeated motif at amino acids 57-I 14 and 21 O-267 in the N-terminal domain of CLIP-l 70. (b) Comparison of CLIP-170 with DP-150 reveals a strong similarity of the repeated motif of CLIP-170 with a motif in DP-150 starting at position 43 in its N-terminal domain and weak similarity between the a-helical domains of the two proteins (amino acids 200-500 and 9501200 in DP-150). (c)Comparison of CLIP-1 70with BlKl, showing the strong similarity of the repeated motif in CLIP-170 with a motif in BlKl starting at position 21 in its N-terminal domain and weak similarity between the a-helical domains of the two proteins (amino acids 200-400 in BIKl). The extreme C-termini of BlKl and CLIP-170 are also very similar.

8wi 700 4 22 c,

I

6W 1

I

500 400

1,

.rw

/ /

200

,

IW

+

/ /

--

-IW

200

300

400

r

SW .6ilo.

700

800

I

,-

900 IO90 1100 1200

TII3W

CUPIM

b

I3W{

‘.





I

1000 900

, /

,

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700.

3w

-

i

.

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CLIP170

,



,

I

Cell 894

a

140 303

121 124 103

b CLIP-170 BlKl GAG Figure

7. Sequence

Alignment

of CLIP-170

with Homologous

Motifs

in DP-150,

Glued,

and BlKl

(a) The repeated motif in the N-terminus of CLIP-170 (rep1 and rep2) and the homologous motifs in DP-150, Glued, and BlKl are aligned. The boxes indicate amino acid identities or conservative changes in at least three of the proteins. (b) The sequence of the C-terminus of CLIP-170 is aligned with homologous motifs in BlKl and HIVl-GAG. Boxes indicate amino acid conservation in at least two of the aligned proteins. Numbers at the right are for the last residue in the row.

proteins (Rickard and Kreis, 1991). Calculation of the native molecular weight of CLIP-170 from these values according to the Svedberg equation (Bloom et al., 1988) gives avalue of 310,000. This corresponds to a dimerized form of CLIP-170, in agreement with the putative coiledcoil structure of the protein. Based on these data, dimensionsof IlOnm by2.5nm wereestimatedfortheCLIP-170 homodimer (Table 1). The comparison between CLIP-170 and BIKl also revealed aconserved motif (Berlinet al., 1990)at thec-terminus (Figure 6~). This “finger” motif of 14 aa, which may form a metal-binding site (Berlin et al., 1990) is distinct from the “zinc finger” motif found in transcription factors (Berg, 1986). It is also found in the small nucleic acidbinding proteins encoded by the gag genes of retroviruses (HIVl, FeLV; Berg, 1986). The consensus sequence of this potential metal-binding motif, CX,CXSGHX& (amino acids 1373-l 386 in CLIP-1 70) is absolutely conserved in CLIP-170, BIKl, and HlVl-GAG as shown in the alignment in Figure 7b. The function of this potential metalbinding motif is unknown. Characterization of the Microtubule-Binding Site of CLIP-1 70 We used an in vitro assay and construction of defined mutants to locate the microtubule-binding site(s) in CLIP170. The cDNA of CLIP-170 was mutagenized either by truncations, internal deletions, or point mutations, and the wild-type and mutant constructs were transcribed and translated in vitro. Binding of the in vitro translated products to microtubules was assayed by cosedimentation with taxol-polymerized tubulin (Rickard and Kreis, 1991). Figure 8a summarizes the constructs used and their microtu-

bule binding activities; Figure 8b shows representative autoradiograms of the microtubule binding assays. For maximal binding of the wild-type construct to microtubules, it was necessary to add apyrase immediately after the translation to deplete the system of ATP. This depletion of ATP presumably prevents phosphorylation of in vitro translated products (phosphorylation of CLIP-170 inhibits its binding to microtubules; Rickard and Kreis, 1991). In vitro translated CLIP-170 binds efficiently to microtubules under these conditions (Figure 8) and all microtubule binding assays were therefore performed in the presence of apyrase. C-terminal truncations of CLIP-1 70 retaining the first 667 aa (data not shown) or the first 346 aa (mutant H; Figure 8) indicate that the basic N-terminal “head” domain binds to microtubules, whereas a mutant with a deletion in the N-terminus does not bind to microtubules (CAH; Figure 8). Thus, we conclude that the basic N-terminal domain of CLIP-1 70 is necessary and sufficient for microtubule binding. This result is consistent with the finding that basic domains of the microtubule-associated proteins MAP2 (Lewis et al., 1988) tau (Lee et al., 1988) the Drosophila 205 kd MAP (Irminger-Finger et al., 1990), MAP4 (Aizawa et al., 1990; West et al., 1991) and MAPlB (Noble et al., 1989) are important in the interaction of these proteins with microtubules. To test whether the N-terminal repeated motif shared by CLIP-1 70 and the other proteins with microtubule-related functions could be involved in the microtubule binding, mutations within this motif were carried out. Effectsof deletions in the N-terminal domain were tested using both the complete (C) and the “head” (H) constiucts. Deletion of the more N-terminal repeat (Rl) from the full-length con-

CLIP-170 695

Links

Endosomes

to Microtubules

PROTEIN SUE

a CI.lP.,,O CR,* cRP c

R,*,It*

l

157 kDa

+

157 kDa

+

157 kh

+

IS7 kDa

CARRI CAR”R**

cm*

I

+I-

149 kDa

+

149 kDa

-+-I-

132 kDa

CAB

MT-BINDING

+/-

122 kDa

35 LO-a

+

35 kDa

(+)

35 kDa

+/-

35 kDa 27 kDa

(+)

27 kDa

A

CLIP-170

B

C Rl*/R2*

CAR1

C

D

CAH

E

H HR2’

F

G

H AR1

H

H Rl*/R2*

I

H Rl*/RZ*

Figure

6. Characterization

of the Microtubule-Binding

Site of CLIP-170

(a) The mutated constructs of CLIP-170. their molecular mass, and their microtubule binding properties are shown. The filled boxes indicate the repeat motifs (Rl, R2), either wild type (black) or mutated (striped). Deletions (A mutants) are indicated with connecting lines. Full-length proteins (C) or head domains only (H, lacking the 1046 C-terminal amino acids) are shown. Binding to microtubules is indicated in the following way: + indicates 100% binding, -indicates 0% binding, +I- indicates 50% binding, and (+) indicates 50%-100% binding. (b) Representative gels from the microtubule binding assays are shown with supernatants (S) and pellets (P) after incubation of in vitro translated products and centrifugation at 20,006 rpm with (+) or without (-) microtubules. (A-H) Autoradiography. (I) Coomassie staining of the same gel as shown in (H) to demonstrate pelleting of the tubulin. Only the relevant molecular size regions of the gels are shown. The names of the mutants analyzed are shown on the right.

Cell 896

struct (CARl) does not affect its binding to microtubules, whereas deletion of the second repeat together with the C-terminal part of the head domain (CAR2) reduces its binding by 50% (Figure 8). In contrast, binding of the head domain alone to microtubules is reduced upon deletion of the first repeat (HARl ; Figure 8). To avoid complications of misfolding of in vitro translated proteins due to large deletions, the sequence PXGKNDG, which is highly conserved in both repeats and in the homologous proteins (Figure 7a), was changed to AXAENDA using point mutations (Rl * or R2’). Mutagenesis of only one of the motifs does not affect the binding of the full-length mutant proteins to microtubules (CR1 * or CR2*), but reduces the binding of the mutated head domain (HRl or HR2”; Figure 8). Mutagenesis in both repeats decreased the microtubule binding efficiency by 50% in the full-length construct (CR1 ‘/R2’) and abolished binding of the head domain to microtubules (HRl */R2*). Each of the repeat motifs seems to contribute independently to the microtubule binding of CLIP-1 70. The involvement of the second motif in the binding of CLIP-170 to microtubules is demonstrated in mutants in which the first repeat was deleted and the second disrupted by point mutations (compare mutants AR1 and ARl/R2*; Figure 8). The role of the first motif in microtubule binding is inferred from the fact that CAR2, but not CAH, cosedimented with microtubules (Figure 8). The two motifs, however, appear todiffer in the efficiency of their microtubule binding, since mutation of the second motif (HR2’) decreases the binding to a greater extent than mutations in the first repeat (HRl *, HARl). As the sequence of the two motifs is almost identical (Figure 7a), the environment of the two motifs might play a role in modulating binding efficiency. The inhibitory effect of point mutations on binding to microtubules is also stronger in the truncated proteins than in the full-length mutants. This difference might be attributable to dimerization of the full-length construct, allowing cooperative binding to the tubulin polymer even with weakened binding sites. Dimerization is unlikely for the head domain alone, since it is lacking the sequence predicted to form a coiled coil, and mutations in its microtubule-binding sites would therefore be more effective. l

Discussion We have established that CLIP-1 70 is involved in binding of endocytic vesicles to microtubules in vitro. In vivo, CLIP-170 is distributed in patches throughout the cytoplasm of HeLa cells. The colocalization of some of these patches with TFR-positive endosomes, especially during BFA-induced microtubule-dependent tubularization of endosomes, is consistent with a role for CLIP-1 70 in the interaction of endocytic organelles with microtubules in vivo. The characteristics of binding of CLIP-170 to microtubules (Rickard and Kreis, 1990) correlate with the characteristics of interaction of endocytic carrier vesicles with microtubules (&heel and Kreis, 1991). CLIP-1 70 and the cytosolic activity promoting binding of endocytic carrier vesicles to microtubules are enriched in a fraction of nucleotide-sensitive microtubule-binding proteins. CLIP-

170 is not tightly associated with kinesin or cytoplasmic dynein, both of which are not required for binding of endocytic carrier vesicles to microtubules. The effects of ATP and AMP-PNP on the binding to microtubules of endocytic carrier vesicles and CLIP-170 are also similar, consistent with the regulation of binding of CLIP-170 to microtubules by phosphorylation (Rickard and Kreis, 1991). The inhibitory effect of GTP on the binding of endocytic carrier vesicles to microtubules (Scheel and Kreis, 1991) is not yet understood, since CLIP-170 contains no known GTPbinding motif. A possible explanation for these GTP effects may be the conversion of GTP to ATP by cytosolic enzymes, or the utilization of GTP by a kinase (Pinna, 1990) phosphorylating CLIP-1 70. Alternatively, since cytosolic and membrane-associated factors in addition to CLIP-l 70 are required for the binding of endocytic vesicles to microtubules, GTP could act on one of these factors. Our analysis of the microtubule-binding region of CLIP170 indicates that one motif per molecule is sufficient for binding to microtubules. On this basis, DP-150, Glued, and BIKl may have the ability to bind to microtubules via this conserved motif present in their basic N-terminal domains. It is not clear, however, why these proteins contain only one motif per polypeptide, whereas CLIP-170 has two. Analysis of the microtubule binding activity of DP-150, Glued, and BIKl would help clarify the significance of this homologous motif. Possibly other regions of these proteins are also involved in microtubule association, since BlKl also contains a tau-like microtubule-binding motif in its N-terminus (Berlin et al., 1990). Dynactin, the 150 kd chicken homolog of DP-150 and Glued, lacks the N-terminal basic domain that contains the CLIP-170 microtubulebinding motif in DP-150 and Glued, which is consistent with the failure of purified dynactin to bind to microtubules (Gill et al., 1991). Since the cloned dynactin is the smallest of several isoforms (Gill et al., 1991) larger isoforms containing the basic N-terminal domain found in the rat and Drosophila homologs might exist and arise by differential processing of the dynactin mRNA, thereby modulating the interaction of dynactin isoforms with microtubules. We have noted a high homology between the putative N-terminal microtubule binding region of DP-150 and the predicted amino acid sequence found in the 5’nontranslated region of dynactin that lacks a stop codon (Gill et al., 1991); this could further indicate generation of dynactin isoforms by differential processing of its mRNA. The overall domain organization of all the proteins sharing the motifs involved in microtubule binding of CLIP-170 is remarkably similar. Although the molecular weight of BlKl is considerably smaller (SO,OOO), it contains a sequence homologous with the microtubule-binding motif of CLIP-170, a central predicted coiled-coil domain, as well as the predicted metal-binding domain (Berlin et al., 1990) that is homologous to the C-terminus of CLIP-170. Since metal-binding motifs are involved in protein-protein, as well as protein-DNA, interactions (Luisi et al., 1991) they could mediate interaction with other components, for example a receptor on the membranes of endocytic vesicles. Considering the evolutionary distance between human and yeast, the high level of conservation of the domains

&P-l70

Links

Endosomes

to Microtubules

and the overall similarity in the structural organization of CLIP-170 and BlKl suggest that these two proteins have related functions. CLIP-170 has no known nucleotide-binding motifs and no similarity to the motor domain of kinesin, which makes it very unlikely that CLIP-1 70 is a motor protein. The molecular design of CLIP-170 is, however, strikingly similar to the kinesin superfamily (Vale and Goldstein, 1990) and to some myosins (Cheney and Mooseker, 1992). This headand-tail organization has been implicated in the movement of various substrates along cytoskeletal structures (Schliwa, 1989; Vale and Goldstein, 1990). A similar design of an elongated molecule, containing a conserved microtubule-binding region at one end and a variable domain at the other end involved in specifying interactions with organelles, could also be used by nonmotor proteins such as CLIP-170, which are involved in linking cytoplasmic organelles to microtubules. A 150 kd protein, presumably homologous to DP-150 and dynactin, has also been observed in HeLa cells (Rickard and Kreis, 1990) suggesting that different proteins with homologous microtubule-binding motifs may perform distinct yet similar functions in the same cell. This suggestion would be consistent with the observed specificity of CLIP-170 for endocytic vesicles but not trans-Golgi network-derived vesicles. CLIP-1 70 may have a role in endocytosis distinct from that of the motor proteins, facilitating the capture of peripheral endosomes by microtubules and allowing their subsequent translocation by motor proteins. This function for CLIP-170 may explain its preferential accumulation at the peripheral plus ends of microtubules in vivo (Rickard and Kreis, 1990). Microtubule plus ends are, however, highly dynamic (for a review see Gelfand and Bershadsky, 1991) and CLIP-1 70 might be involved in their capture and stabilization to regulate their dynamics (Kirschner and Mitchison, 1986). Stabilization of microtubules with taxol induces bundling of microtubules into the cell center and also leads to an accumulation of CLIP-170 at the cell periphery (Rickard and Kreis, 1990). This may reflect the formation of endocytic organelles that cannot interact with microtubules since they no longer extend all the way to the cell periphery. In this respect, it is notable that CLIP-170 has also been localized at desmosomal plaques at the plasma membrane of polarized epithelial cells (Wacker et al., 1992). Interaction of microtubules with peripheral organelles via CLIP-1 70 may therefore reflect a general mechanism for transient stabilization of peripheral dynamic microtubule plus ends, which can be exploited to allow more permanent stabilization of microtubules such as occurs during epithelial cell polarization (Pepperkok et al., 1990). Thus, CLIP-170 may act as a general linker, establishing initial contact of endocytic carrier vesicles and assembling desmosomes and perhaps other structures with microtubules. Binding of CLIP-170 to microtubules in vitro is inhibited by phosphorylation of CLIP-1 70 (Rickard and Kreis, 1991). CLIP-170 is heterogeneously phosphorylated in vivo (J. E. Ft. and T. E. K., unpublished data), and its in vivo phosphorylation state is affected by microtubule-active drugs (Rickard and Kreis, 1991). Phosphorylation at dis-

tinct sites may be involved in regulating its interactions with organelles in addition to microtubules, and sequential phosphorylations could be controlled by sequential interactions of CLIP-170. For example, capture of endocytic carrier vesicles by microtubules via CLIP-170 may be followed by phosphorylation to weaken the microtubule interaction and allow their movement along microtubules by motor proteins. Further experiments using protein expressed in vitro to examine the domains of CLIP-170 involved in its interaction with endocytic carrier vesicles and the regulation of binding to membranes and microtubules should now be possible using in vitro assays. Identification of the functional and regulatory domains of CLIP-170 will then allow an analysis of the activity and function of this protein in vivo. Experimental

Procedures

Antibodies The MAb 2D6. which belongs to the IgM subclass, was obtained in the same fusion as MAbs 403 and 3A3 (Rickard and Kreis, 1991). SUK4 was a gift of Jonathan Scholey (Ingold et al., 1968). lmmunoglobulin fractions of 3A3, 2D6, and SUK4 were prepared from ascites and coupled to CNBr-activated Sepharose (Pharmacia-LKB) as previously described(Rickard and Kreis, 1991). Peptideantibodieswere raised by immunization of rabbits and affinity purified from serum as described (Duden et al., 1991). Polyclonal antibodies against CLIP-170 were obtained by immunization of a rabbit with bacterially expressed fusion protein of clone 55 in ptJEX and affinity purified on blots (Rickard and Kreis, 1990). Preparation of Cytosol, Endocytic Carrier Vesicles, and CLIP-l 70 Cytosol from HeLa cells and HRP-labeled endocytic carrier vesicles were prepared as described (Scheel and Kreis. 1991). In brief, endocytic carrier vesicles were labeled by internalization of HRP in nocodazole-treated baby hamster kidney cells (Gruenberg et al., 1989). Sodium chloride, at a concentration of 1 M, was added to a postnuclear supernatant prepared from these cells, and a fraction enriched in saltwashed endocytic carrier vesicles was obtained by flotation in a sucrose gradient. HeLa cytosol was prepared and pretreated with antibodybeadsasdescribed(Scheeland Kreis, 1991). Tofractionate HeLa cytosol by ammonium sulfate precipitation, solid ammonium sulfate was added to 40% saturation to the cytosol and the suspension was incubated on a rotating wheel for 1 hr at 4°C. Precipitated proteins were removed by centrifugation for 10 min at 10,000 x g at 4% and the supernatant was dialyzed against 0.1 M PIPES-KOH, 1 mM EGTA, 1 mM MgSOI (pH 6.8). CLIP-l 70 was isolated from HeLa cytosol as described (Rickard and Kreis, 1991) with the following changes. ATP was not depleted in the cytosol and Triton X-100 was omitted from the washing and elution steps. After binding of CLIP-170 to MAb 3A3 on Sepharose beads and subsequent washing of the beads, 20 ul beads were packed in a 200 ul pipette tip (Gilson). To elute CLIP-170 from the antibody, 50 ul of 0.1 M triethanolamine, 1 M NaCl (pH 11.5) was passed through the tip and directly pipetted into 20 ul of 1 M PIPES-KOH (pH 6.8). The eluate was dialyzed against 0.1 M PIPES-KOH, 1 mM EGTA, 1 mM MgSOI (pH 6.9). In Vitro Organelle Binding Assay Binding of endocytic carrier vesicles to microtubules was measured asdescribed(Scheeland Kreis, 1991). In brief, taxol-stabilizedmicrotubules from bovine brain were coupled to magnetic beads. Salt-washed endocytic carrier vesicles labeled with HRP were incubated with the microtubule beads and cytosol preparations for 50 min at 23°C (Scheel and Kreis, 1991). The magnetic microtubule beads with bound organelles were retrieved with a magnet and the HRP activity was measured (Gruenberg et al., 1989) in the bound and unbound fractions.

Cell 898

lmmunotluorescence Microscopy HeLa cells fixed for 4 min in methanol at -20% were labeled with affinity-purified rabbit antibodies to CLIP-170 (Rickard and Kreis, 1990) and a mouse MAb against human TFR (Boehringer Mannheim) followed by rhodamineand fluorescein-labeled secondary antibodies (Rickard and Kreis, 1990). Samples were mounted in mowiol and examined by conventional fluorescence microscopy or mounted in 50% glycerol in phosphate-buffered saline containing 100 mglml 1,4diazabicyclo-(2,2,2)octane as antifading agent and exammed on the modular confocal microscope constructed at the European Molecular Biology Laboratory as described (Wacker et al., 1992). Stereo pairs were calculated from optical sections recorded at 0.4 urn vertical steps. Protein Sequencing Approximately 100 ug of affinity-purified CLIP-170 (Rickard and Kreis, 1991) was run on a preparative 7% SDS-polyacrylamide gel. The CLIP-170 band stained with Coomassie blue was excised, washed with water, and digested for 12 hr with trypsin (10% [w/w]; sequencing grade; Boehringer Mannheim). Peptides were separated by reversephase high pressure liquid chromatography on a microbore column and eluted with a gradient of O%-80% acetonitrile in 0.1% aqueous trifluoroacetic acid. Fractions containing peptides were lyophilized and sequenced as described (Duden et al., 1991). Isolation of cDNA Clones A mouse antiserum raised against ATP-sensitive microtubule-binding proteins of HeLa cells (Rickard and Kreis, 1990) was used to screen approximately 400,000 colonies of a random-primed HeLa cell cDNA library prepared in the expression plasmid pUEX (Bressan and Stanley, 1987). Fusion proteinsof positiveclones were used toaffinity purify antibodies from the serum on nitrocellulose blots (Rickard and Kreis, 1990). Five clones (28, 30, 35, 37, and 55) were isolated on the basis of the specificity for CLIP-170 of antibodies affinity purified on the corresponding fusion proteins; these five clones were shown to crosshybridize by Southern blotting. The insert of clone 55 (2 kb, nucleotides 784-2716 in the full-length construct), labeled with ‘*P using the Random-Primed Labeling kit (Boehringer Mannheim), was used to rescreen the same cDNA library as well as 500,000 clones of each of two HeLa cDNA libraries (kindly provided by Dr. P. Chambon, Strasbourg) in the vector hZAPll (Stratagene. LaJolla, California): one prepared from random-primed cDNA; the other prepared with oligo(dT)primed cDNA. After transfer of bacterial colonies or phages onto nitrocellulose (Sambrook et al., 1989) the filters were prehybridized for 3 hr at 68% in 6x SSC (1 x SSC is 0.15 M NaCl plus 0.015 M sodium citrate), 5x Denhardt’s solution, 0.1% SDS, 10 mM EDTA, 100 uglml herring sperm DNA. Hybridization was performed overnight with the radioactive probe in the same solution at 68°C. Filters were washedin x SSC, 0.1% SDSfor 15min at room temperaturefollowed by two washes in 0.1 x SSC, 0.1% SDS at 68V for 30 min, air dried, and exposed to X-ray film (X-Omat AR; Eastman Kodak, Rochester, New York). Forty-five clones were isolated from the three cDNA librarres and characterized by restriction mapping and Southern blotting. Clone Ml (2.5 kb, nucleotides 122-2643 in the full-length clone) from the pUEX library, clone 8A2 (3.3 kb, nucleotides 507-3570) from the oligo(dT)-primed hZAPll library, clone RDlA(2.4 kb, nucleotides27355161) from the random-primed lZAPll library, and clone Hi (2.4 kb, nucleotides 1-1451) from the pUEX library were analyzed in detail. DNA Subcloning and Sequencing DNA isolation from transformed bacteria and recombinant DNA manip ulations were performed using standard procedures (Sambrook et al., 1989). The inserts of the pUEX clones were subcloned into the plasmid Bluescript KS (Stratagene). Clones obtained from the hZAPll libraries were excised as Bluescript KS plasmid with helper phage according to the manufacturer’s instructions, and plasmid DNA was analyzed further. After restriction mapping, five clones were selected (clones Hl, Ml, RDl A, 8A2, and 55) for DNA sequencing. Unidirectional deletions of the clones were made using a Nested Deletion Kit (PharmaciaLKB, Freiburg, Germany). Plasmid DNA, purified on Quiagen columns (Diagen GmbH, Dusseldorf, Germany), was used for dideoxynucleotide sequencing with [a%]dATP and a T7 sequencing kit (PharmaciaLKB) or with fluorescent primers and the European Molecular Biology Laboratory sequencing device. The clones were sequenced on both

strands and were identical in the regions of overlap. To fill the remaining gaps in the sequence, synthetic oligonucleotides were used as sequencing primers to obtain overlapping sequences on both strands, Sequence data were compiled and analyzed using the UWGCG package as described by Duden et al. (1991). The full-length cDNA clone of CLIP-170 was assembled in Bluescript KS using four different clones (Hl, Ml, 8A2. and RDl A). First, clone 8A2 and RDlA were assembled using the Xhol site and ligated in the plasmid at the BamHl and EcoRl sites (plasmid ~~1704). Clone Ml or Hl containing the ATG initiation codon was then assembled at sites BamHl and Sphl with the plasmid ~~1704 to create the full-length open reading frame (plasmid pMlCLIP-170 and pHlCLIP-170; Figure 3). All in vitro transcription and translation reactions were performed with the Ml form of CLIP-170, which gave a better translation efficiency than the Hl form. Sequence Analysis The secondary structure prediction, the molecular size, and the amino acid composition of CLIP-170 were established using the GCG programs PEPTIDE-STRUCTURE and PEPTIDESORT (Devereux et al., 1984). FASTA (Pearson and Lipman, 1988) and BLAST (Altschul et al., 1990) were used to search the GenEMBL nucleotide data library and the SwissProt protein data library for sequences homologous to CLIP170. Homologies were analyzed using the Sensitive Sequence Comparison program (ISSC; Rechid et al., 1989). Sequence alignment was performed using the PILE-UP and PRETTYPLOT programs. Potential sites for posttranslational modifications were identified by searching the PROSITE data library (Bairoch, 1989) with the program SCRUTINEER (Sibbald and Argos, 1990). Generation of Mutant cDNA Clones Mutant CAR1 (amino acids 55-124 deleted) was created by replacing the BamHI-Hpal fragment (nucleotides 30-537) of pMlCLIP-170 by a BamHI-Hpal fragment containing the cDNA sequence from nucleotides l-310. This fragment was generated by a polymerase chain reaction procedure on clone Ml using the Bluescript universal primer and the oligonucleotide S-TGTGTTAACTCCACAAATTCCTCCTGAGTGTG-3’ containing nucleotides 310-290 of pCLIP-170 and an Hpal site. Mutant CAH (amino acids 55-346 deleted) was constructed using the plasmid pCARl[A55-1241, which was digested at the Hpal and Bglll sites and religated after filling of the Bglll overhang with Klenow enzyme and d-nucleoside triphosphate. Mutant CAR2 (amino acids 124-346 deleted) was constructed as described for CAH using pCLIP-170 instead of pCAR1 [A55-1241. Point mutations were created in the cDNA by using specific oligonucleotides in an approach based on polymerase chain reaction (Horton et al., 1989) and recloning the mutagenized fragments using BamHl and Nhel restriction sites. All the mutants created by polymerase chain reaction were sequenced to control for the absence of nonspecific mutations. In Vitro Transcription and Translation The different Bluescript templates were linearized at convenient restriction sites (EcoRI or Bglll) and RNAs were produced by in vitro transcription using T7 polymerase and an in vitro transcription kit (Promega) according to the manufacturer’s instructions. RNA was translated using rabbit reticulocyte lysate (Promega) for 60 min at 30% using [%]methionine (Sambrook et al., 1989). Translation products were analyzed by SDS-polyacrylamide gel electrophoresis and fluorography (Scheel et al., 1990). In Vitro Microtubule Binding Assay Ten microliters of 100 mM methionine and 2 1.11of 200 U/ml apyrase were added to 28 ul of the in vitro translation reactions and incubated for 10 min at 30°C. Eighty microliters of PEM buffer (100 mM PIPESKOH. 2 mM EGTA, 1 mM MgCI,, 2 mM dithiothreitol [pH 6.81) plus 20 uM taxol was added and the samples were spun at 55,000 rpm (100,000 x g) for 1 hr at 30°C in a TLA-100 rotor (Beckman Instruments, Palo Alto, California). The supernatant was split in two and incubatedat30°Cfor30min withorwithout30 ugoftaxol microtubules (Rickard and Kreis, 1991). The samples were layered over 100 ul of 30% sucrose in PEM and centrifuged at 20,000 rpm (30,000 x g) in an SW50.1 rotor (Beckman Instruments) for 30 min at 30%. Supernatants and pellets (washed once with PEM buffer) were analyzed by SDS-

CLIP-170 899

Links

Endosomes

to Microtubules

polyacrylamide gel electrophoresis and fluorography. All gels were stained with Coomassie blue to verify that added taxol-polymerized tubulin was quantitatively recovered in the pellet. Physical Characterization of CLIP-170 The sedimentation coefficient, So,,. of CLIP-170 was measured by sucrose gradient centrifugation as described previously (Rickard and Kreis, 1990; Duden et al., 1991) except that the gradients were 5%20% sucrose. Standard proteins and their sedimentation coefficients (x 10j3 s) were horse spleen apoferritin (17.60) bovine liver catalase (11.30) sweet potato 8-amylase (8.98) human fibrinogen (7.63). yeast alcohol dehydrogenase (7.40) and bovine serum albumin (4.41). The diffusion coefficient, Dm.w, was measured by gel filtration chromatography on a Superose 6 fast protein liquid chromatography column (HR 10/30; Pharmacia-LKB) equilibrated with PEM buffer and run at 0.2 ml/min. The sample volume was 0.2 ml. Standard proteins and their diffusion coefficients (x 10’cm2/s) were human fibrinogen (1.98) bovine thyroglobulin (2.50) horse spleen apoferritin (3.61) sweet potato p-amylase (5.77). and bovine serum albumin (6.90). All standard proteins were purchased from Sigma. The native molecular weight, Stokes radius, axial ratio (assuming a molecular weight of 3.1 x 105), and approximate dimensions were calculated according to Bloom et al. (1988). Miscellaneous Restriction enzymes and other molecular biology reagents were purchased from Boehringer Mannheim unless stated otherwise. All radioactive chemicals were obtained from Amersham International (Braunschweig. Germany). Gel electrophoresis of proteins and immunoblotting were performed as described (Rickard and Kreis, 1990). Acknowledgments Thefirsttwoauthorscontributedequallytothiswork. WethankGeorge Banting and Keith Stanley for their help with preparing the HeLa cDNA expression library, Shamsa Faruki and Ed Hurt for total HeLa RNA, and Jonathan Scholey for the MAb SUK4. The random- and oligo(dT)primed lZAPll libraries were generous gifts of Dr. P. Chambon (Strasbourg). We appreciated the help of Pat Argos and Thure Etzold with the structure analysis of CLIP-170. We are grateful to our colleagues at European Molecular Biology Laboratory for many stimulating discussions during the progress of this work and particularly to Spyros Georgatos, Jean Gruenberg, Brian Storrie, and John Tooze for their valuable comments on the manuscript. We acknowledge Brigitte Joggerst-Thomalla for excellent technical assistance. Taxol was obtained from Dr. N. Lomax (Department of Health and Human Services, National Institutes of Health, Bethesda, Maryland). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 USC Section 1734 solely to indicate this fact. Received

April 27, 1992; revised

July 7, 1992.

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cytoplasmic

Accession

The accession M97501. Note Added

number

Brefeldrn network

A causes and early

Number for the sequence

reported

in this paper

IS

in Proof

Recently, the sequence of another protein of unclear function, identical to CLIP-170 except for a 35 aa insert, was published (X648381; Bilbe et al., EMBO J. 71, 2103-2113, 1992).

CLIP-170 links endocytic vesicles to microtubules.

Binding of endocytic carrier vesicles to microtubules depends on the microtubule-binding protein CLIP-170 in vitro. In vivo, CLIP-170 colocalizes with...
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