Isolation and sequencing of a cDNA clone encoding 96 kDa sialoglycoprotein in rat liver lysosomal membranes

Vol. 164, No. 3, 1989 November

BIOCHEMICAL

AND BIOPHYSICAL

15, 1989

RESEARCH COMMUNICATIONS Pages 1113-1120

Isolation and sequencing of a cDNA clone encoding 96 kDa sialoglycoprotein lysosomal membranes

in rat liver

Youichiro Noguchi, Masaru Himeno, Hiroyuki Sasaki+, Yoshitaka Tanaka, Akira Kono*, Yasuyuki Sakaki+, and Keitaro Katol Division of Physiological Chemistry, Faculty of Pharmaceutical Sciences, Kyushu University, Higashi-ku, Fukuoka, Japan +Research Laboratory for Genetic Information, Kyushu University, Higashi-ku, Fukuoka, Japan *National Kyushu Cancer Center, Minami-ku, Fukuoka, Japan Received

September

22,

1989

SUMMARY: We isolated and sequenced LGP 96, a cDNA clone corresponding to the entire coding sequence of the rat liver lysosomal membrane sialoglycoprotein with an apparent Mr of 96 K, LGP 96. The deduced amino acid sequence indicates that LGP 96 consists of 411 amino acid residues (Mr 45,163) and the 26 NH2-terminal residues presumably constitute a cleavable signal peptide. The major portion of LGP 96 resides on the luminal side of the lysosome and bears a large number of N-linked heavily sialylated complex type carbohydrate chains, giving the mature molecule of 96 kDa. The protein has 17 potential N-glycosylation sites and 32.1 and 65.3% sequence similarities in amino acid to LGP 107 and human lamp-2,respectively. The glycosylation sites are clustered into two domains separated by a hinge-like structure enriched with proline and threonine. LGP 96 possessesone putative transmembrane domain consisting of 24 hydrophobic amino acids near the COOH-terminus and contains a short cytoplasmic segment constituting 12 amino acid residues at the COOH-terminal end. Comparison of LGP 96 and recently cloned lysosomal membrane glycoprotein sequences reveals strong similarity in the putative transmembrane domain and cytoplasmic tail. It is very likely that these portions are important for the targeting of molecules to lysosomes. A comparison of LGP 96 and LGP 107 showed numerous structural similarities. 0 1989 Academic PEES.inc. Lysosomal hydrolases are delivered to lysosomes or prelysosomes from the trans-Golgi network (TGN) in which the enzymes are bound to an integral membrane protein carrier, the mannose-6-phosphate receptor (MPR), that is capable of functioning in multiple cycles of hydrolase delivery (1,2). On the other hand, lysosomal membrane glycoproteins are brought to lysosomes independent of MPR from TGN, by unknown mechanisms (3-5). Numerous lysosomal membrane glycoproteins have been purified from various species (6-14), however, functions and mechanisms of intracellular transport of these glycoproteins remain unknown. For elucidation, it is essential to isolate and characterize individual lysosomal membrane glycoproteins and to clone their cDNAs. We purified the major sialoglycoproteins, LGP 107 and ‘To whom correspondence should be addressed at Division of Physiological istry, Faculty of Pharmaceutical Sciences, Kyushu University, Higashi-Ku, Fukuoka 812, Japan.

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Copyright 0 I989 All rights of reproduction

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Vol. 164, No. 3, 1989

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LGP 96, from the rat liver lysosomal membranes and a cDNA for LGP 107 was isolated and sequenced (9). Here we report the isolation and sequencing of a cDNA clone encoding LGP 96. Comparison of the deduced amino acid sequence of LGP 96 with that of LGP 107 indicates that rat LGP 96 and LGP 107 share common structural features (32.1% similarity). The deduced amino acid sequence of LGP 96 was also compared to the human lamp-2 (h-lamp-2) (15) sequence. As LGP 96 more closely resembles h-lamp-2 (65.3% similarity) than LGP 107, LGP 96 and h-lamp-2 probably belong to the same gene family in different species. MATERIALS

AND METHODS

Purification of LGP 96from rat liver lysosomal membranes

The major sialoglycoproteins (420 kDa) were purified from rat liver lysosomal membranes, as described (8,9). When analyzed by SDS-PAGE, the purified proteins were separated into two polypeptide bands with molecular masses of 96 (LGP 96) and 107 kDa (LGP 107). The two polypeptides, separated by preparative SDS-PAGE, were extracted from the gel and used as purified preparations. Antibodies against LGP 96

Antibodies were raised in rabbits against the purified LGP 96 and affinity purified on a Sepharose 6B column conjugated with purified LGP 96, as described (9). Protein sequence analysis

The NI-IZ-terminal sequence of purified LGP 96 was determined in an Applied Biosystem 470 A protein sequencer/Spectra Physics SP8100 HPLC system. Screening of the cDNA library

A rat liver cDNA expression library (16) in hgtll was screened with specific rabbit antibodies raised against LGP 96, using horseradish peroxidase-conjugated anti-rabbit IgG as the second antibodies, as described (17). A positive plaque (Ll) was isolated and the bacteriophage DNA was digested with EcoR I. The EcoR I-excised cDNA insert was subcloned into plasmid vector pUC118 (18), and characterized by restriction-endonuclease mapping. To obtain longer cDNAs of LGP 96, we prepared an EcoR I-Pst I fragment (5 11 bp) from the subcloned cDNA and screened the library with the EcoR I-Pst I fragment as a probe. Four positive clones were obtained. The one (L2) with the longest cDNA insert was analyzed further. DNA sequencing

Restriction endonuclease fragments of cDNA of L2 were subcloned into plasmid vector pUC118 (18). Single stranded DNAs, isolated with the aid of helper phages (M13K07), were sequenced by the dideoxynucleotide chain termination methods (19) using the SequenaserM DNA-sequencing kit. Computer analysis of cDNA and protein

Nucleotide and protein sequences were analyzed using the GENAS System at Kyushu University Computer Center (20). Hydropathy analysis was performed according to Kyte and Doolittle (21). RESULTS AND DISCUSSION Isolation of cDNA clones

A rat liver cDNA library constructed with hgtl 1 as vector (16) and screened with anti-LGP 96 antibodies led to isolation of single positive clone from approximately 10s phages. The clone was termed Ll, (insert length, 1,239 bp). The inserted cDNA fragment was subcloned into the plasmid vector pUC118 and sequenced. At a result of the sequencing, the cDNA did not contain codons corresponding to the N-terminal amino acid residues that we sequenced from purified 1114

Vol. 164, No. 3, 1989

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LGP 96 but did show a close similarity to the downstream of h-lamp-2 cDNA (15). Therefore, the EcoR I-Pst I cDNA fragment with 511 bp was prepared from the Ll clone, labeled with 32P, and used as a probe to isolate the cDNA clones containing much longer insert from this library. We finally obtained 4 positive clones that have longer cDNA inserts than that of the Ll clone. Sequence analyses of the cDNA and structure of LGP 96 The longest cDNA fragment (L2; 1,547 bp) was subcloned into the plasmid vector pUCll8 and analyzed by restriction mapping.

Fig. 1 shows the restriction map of the insert of L2, the

cDNA for LGP 96, as well as the strategy adopted for sequencing. Fig. 2 shows the nucleotide sequence determined from the L2-cDNA

and the deduced primary structure of LGP 96. As

shown in Fig. 2, composition of the L2-cDNA sequence has an open reading frame of 1,236 bp (nucleotides 93-1,328) flanked by 92 nucleotides of 5’- and 220 of 3’-untranslated sequence. This clone has no poly(A) tail. Therefore, complete 3’-untranslated region is probably much longer. The NHZ-terminal sequence determined from purified LGP 96, Leu-Lys-Leu-X-Leu-ThrAsp-Ser-Lys-Gly-Thr-X-Leu-Tyr-Ala-Glu-Tr-Glu-Met-,

was identical to the NH2-terminal

sequence predicted from the nucleotide sequence of L2-cDNA. Thus we concluded that this clone has the cDNA insert for the entire translating region of LGP 96. The LGP 96 polypeptide comprised 411 amino acid residues (Mr 45,163), including NH2-terminal26

amino acid residues

that constitute a cleavable signal peptide, because positions -3 and - 1 relative to the cleavage site conform to “-3, -1 rule” as suggested by von Heijne (22, 23). LGP 96 has 17 potential Nglycosylation sites, all of which are probably utilized in the mature protein, judging from the molecular difference between the primary structure deduced from the nucleotide sequence and the mature one. Eight cysteine residues were included, 4 of which were uniformly spaced at intervals of 32-36 residues and three at intervals of 47, 65 and 73, respectively.

The primary

structure of LGP 96 contains a potential hinge region (residue 196-225) that is rich in proline and

Ham

HI

Bin

dII1

Pvu

II

Bin

cI1

Fig. Restrictionmap of LGP 96 cDNA and the strategyadaptedfor nucleotide sequence determination. Arrows indicate the dire&on and extent of sequencingruns. The open box indicatesthe amino acid coding region and the lines indicate the 5’-and 3’-noncoding regions. 1115

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GT CGG TGG TCG CCC GTG CTT KG CTT TCT CAG GGC TGT GAG GGT GTT CGT TGG AAT TGT CGG TCC AGT CGT CAC TTG TCC TGA GGG ATC ACG ATG

Met 96 CGC CTT CCT CTC XC 2 _Ars -------Leu Pro Leu Ser

GGT TAC GGC TCG RAG CTC GTC CTG CTC TTT CTG Gly Tyr G~J -- SerL~s -----------Leu "al Leu Leu Phe Leu

144 TTC CTG GGA GCA GTT CGG TCC 18 ----Phe Leu GlrAla---- Val Ag ---Ser 192 34 240 50

GAT GCA TTG As2 --Ala Leu t TCA AAG GGT ACT TGC CTT TAT GCA GAA TGG Ser Lys Gly Thr Cys Leu Tyr Ala Glu Trp # ACA TAT GAA GCT CTA AAA GTC AAT GAA ACT Thr Tyr Glu Ala Leu Lys Val Asn Glu Thr

AAA CTT AAT Lys Leu As* * GAG ATG AAT Glu Met Asn

TTG ACA GAT Leu Thr Asp TTC ACA ATA Phe Thr Ile

l

GTA ACC ATT ACA GTG CCT Val Thr Ile Thr Val Pro

l

288 66 336 82

GAC AAG GTG ACA TAC AAT GGA AGC Asp Lys Val Thr Tyr Asn Gly Ser + GCC AAA ATA ATG ATA CAA TAT GGA Ala LyS Ile Met Ile Gin Ty? Gly

AGT TGT GGC Ser Cys Gly # TCC ACT CTC Ser Thr Leu

GAT GAT AAG AAT Asp Asp Lys Asn

384 98

TTC ACC AAG GAA GCA TCT CAG TAT TTT ATT AAC AAC ATC ACG CTT TCT Phe Thr Lys Glu Ala Ser Gin Tyr Phe Ile Asn Asn Ile Thr Leu Ser

432 114

TAC AAC ACT Tyr AS,, Thr

TCT TGG GCT Ser Trp Ala

GGT Gly

GTG AAT Val Asn

l

480 130 528 146 576 162

AAT GAT ACC Asn Asp Thr * ATC CTT ACT GTT ATC ATT Ile Leu Thr Val Ile Ile

ATC TTT AAG TGC AGT Ile Phe Lys Cys Ser x CAG CAC TAT TGG GGC Gln His Tyr Trp Gly

AAA ACA TTT CCT GGT GCT GTA CCT AAA GGA Lys Thr Phe Pro Gly Ala Val Pro Lys Gly CCT GTG GGA TCC Pro Val Gly Ser

CAG CTT CCA Gln Len Pro

TTG GGT GTC Leu Gly Val

AGT GTT TTA ACT TTC AAC CTG AGT CCT GTT Ser Val Leu Thr Phe Asn Leu Ser Pro Val f ATT CAC CTG CAA GCT TTT GTC CAA AAT GGT Ile His Leu Gln Ala Phe Val Gin Am Gly

GTT Val RCA Thr

l

624 178 672 194

GTG AGT AAA CAT GAA CAA GTG TGT AAG Val Ser Lys His Glu Gin Val CyS LyS # GTA GCA CCC ATC ATT CAC ACC ACC GTG Val Ala Pro Ile Ile His Thr Thr Val -1111111111111111111lllll

GAG GAC AAA ACT GCT ACC ACT Glu Asp Lys Thr Ala Thr Thr CCA TCG CCT ACT ACG ACA CTC Pro Ser Pro Thr Thr Thr Le"

720 210

ACT CCA ACT TCA ATA CCC GTT CC* ACT CCA Thr pro Thr Ser Ile Pro Val Pro Thf Pro 1111111~111111111111~-~-~-~--

768 226 816 242

ATT TCT AAT GGC AAT Ile Ser Asn Gly Asn * CTG AAC ATC ACC GAG Leu Asn Ile Thr Glu

864 258

GCC ACA ACC AAC TTC ACC GGC AGC TGT CAG CCC CAA ACA GCT CAA CTT Ala Thr Thr Asn Phe Thr Gly Ser Cys Gin Pfo Gin Thr Ala Gin Leu

912 274

AGG CTG AAC AAC AGC CAA ATT AAG TAT Arg Leu As" As,, Ser Gl" Ile Lys Tyr

GCT ACC TGT Ala Thr Cys # GAG AAG GTG Glu Lys Val

l

ACG GTT GGA AAC TAC ACC Thr Val Glv Asn Tvr Thr

CTG CTG GCT ACC ATG GGG CTG CAG Leu Leu Ala Thr Met Gly Leu G;n CCT TTC ATT TTT AAC ATC AAC CCT Pro Phe Ile Phe Asn Ile Asn Pro

x

CTC GAC Le" Asp

TTT ATC TTT GCT GTG Phe Ile Phe Ala Val

l

960 290 1008 306

AAA AAT GAA AAA CGG TTC TAT CTG AAG GAA GTG AAT GTC AAC ATG Lys As" Glu Lys Arg Phe Tyr Leu Lys Glu Val Asn Val Asn Met

TAT Tyr

1056 322

TTG GCT AAT Leu Ala Asn * TGG GAT GCT ~rp Asp Ala

1104 338

GTT TCC GTG Val Ser "al

1152 354 1200 370

CAA CCT TTT AAT GTG Gln Pro Phe As" "al * AGT GCA GAT GAA GAC Ser Ala Asp Glu Asp

1248 386

CTG GGA GGA GTA CTT ATT CTA GTG TTG CTG GCT TAT TTT ATT GGT CTC Leu Gly Gly "al Leu Ile Leu Val Le" Leu Ala Tyr Phe Ile Gly Leu

1296 402

AAG CGC CAT CAT ACT GGA TAT GAG CAA TTT TAG CACCTACAATCTGATTGAA Lys Arg His His Thr Gly Tyr Glu Gin Phe ---

1348 1411 1474 1537

GGC TCA GCT TTC CAT GTT TCA AAT AX Gly Ser Ala Phe His Val Ser Asn Am

AAC CTT AGC TTC Am Leu Ser Phe

CCT CTG GGA AGT TCT TAT ATG TGC AAC Pro Leu Gly Ser Ser Tyr Met Cys Asn x TCT CGA ACA TTT CAG ATA AAT ACC TTT Ser Arg Thr Phe Gln Ile Asn Ths Phe

AAA GAG CAG GTG Lys Glu Gin Val

l

AAC CTG AAG GTG AS* Le" Lys Val

ACG AAA GGA GAG TAT TCT ACA GCT CAA GAC Thr Lys Gly Glu Tyr Ser Thr Ala Gln Asp

TGC Cys # AAC TTC CTT GTG CCC ATA GCG GTG GGA GCA GCC As" Phe Leu Val PIO Ile Ala Val Gly Ala Ala

TATATTACRRAATACATACAAATAACAAAGTTGTCTTACCTGTCAGTTTATG-CACTTTGC TACTTAAGACAGACTCCTTGGAACTTTTATTTGGAARTCAGTCCCCAC~TTTGAGAT~GGC AAAAAAATCATTCTCTGATCT CAACAPAAAAACAAAAAAC TTTTTTTGAAAAAAAACA GCTTAAAATGCA

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-r J

Fia. Hydropathy plot of LGP 96 protein sequence.Hydrophobicity values obtained according to Kyte and Doolittle (21) have been plotted with respectto positionsin the amino acid sequence. The window usedin the scanningwas 11 amino acids. Line segmentsabove and below the horizontal axis indicate hydrophobic and hydrophilic portions, respectively. A long arrow indicatesthe signal peptidasecleavagesite for the enzyme.The membrane spanningregion (hatchedbox) and potential N-linked glycosylation sites(smai1arrows) are also indicated on the stick diagram above the hydropathy plot.

threonine and is thought to separate the polypeptide into two domains. From the hydropathy analysis (Fig. 3), the protein has a strong hydrophobic region

in proximity

to the COOH-

terminus and the region was thought to be a putative transmembrane domain (residues 376-399) which showed high similarity among the lysosomal membrane glycoproteins, as shown in Fig. 4. We expect that the remaining short COOH-terminal portion (residues 400-411) is extruded to the cytoplasm as a cytoplasmic tail.

We also suggest from the result obtained by treatment of

tritosomes with neuraminidase that the oligosaccharide chains containing sialic acid in LGP 96 are on the luminal side of the tritosomal membranes (data not shown). Comparison of LGP 96 with LGP 107 We recently isolated a cDNA clone coding the entire LGP 107. The previously cloned cDNA for LGP 107 (9) seems to be missing the signal peptide region.

Comparison of the

deduced amino acid sequence of the newly cloned LGP 107 with that of lgp120 (24), the 120 kDa lysosomal membrane glycoprotein (NRK cells), revealed that both proteins are the same except for one amino acid which is changed from Val to Leu located at -7 position in the newly isolated LGP 107 clone. Comparison of the deduced amino acid sequences between LGP 96 and LGP 107 indicated that LGP 96 and LGP 107 consist of 411 and 407 amino acid residues, respectively, and suggest that 26 and 21 NH2-terminal residues are cleavable signal peptides. The deduced amino acid sequence of LGP 96 showed a 32.1% similarity to that of LGP 107. LGP 96 and LGP 107 were found to contain 17 and 20 potential N-glycosylation

sites,

respectively, and these N-glycosylation sites are clustered into two domains separated by a hingelike structure. The hinge region separating the molecule into two domains is rich in proline and &&. The nucleotide sequenceand deduced amino acid sequenceof LGP 96 cDNA. The ammo acid sequenceobtained from protein sequencingis indicated by the box. The upward arrow indicatescleavagesite for the signal peptide. The putative signal sequenceis indicated by a thin broken underline and hinge region by thick broken underline. Underline indicates a stretch of hydrophobic amino acids,a possibletransmembranedomain. Potential N-glycosylation sites, cysteine,and the termination codon are indicated by *, #I,and ---, respectively. 1117

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BIOCHEMICAL

Name

LGP96 LGP107

Amino

380 390 FLVPIAVGAALGGVLILVLLAYFI

RESEARCH COMMUNICATIONS

% Similarity 400 . .

91%

(22/24)

400

:::::::::::.::::::::::::

h-lamp2

APase

sequence

380 390 FLV-PIAVGAALGGVLILVLLAYFI : : : : : .:' : .LIPIAVGGA,,G,;,;G,;,,Li " 380 390

LGP96

LGP96

acid

AND BIOPHYSICAL

100%

(24/24)

FLWIAVGAALAGVLILVLLAYFI 380 390 380 390 FLVPIAVGAALAGVLILVLLAYFI .:..:: . . . .:..::. TEVIVALAVCGSILF&VLLLTV-LFR 390 400

400 .

75%

(18/24)

Fi ,4. Comparisonof amino acid sequencesimilarity of transmembranesegmentof LGP 107, hl-5 amp and acid phosphatase(APase)to that of LGP 96. The transmembranesequenceof LGP 96

(upper) and the counter part of each protein (lower) are representedin the standardone-letter code. The right side numbers show the similarity in %. Numbers shown above and under the one letter coded amino acidsindicate the sequencenumbers of each protein. The same amino acid residuesare indicated by (:), identical residuesby (.), gaps by (-).

threonine in LGP 96 but that in LGP 107 is enriched with proline and serine. Both proteins contained 8 cyteines regularly spaced in these molecules, suggesting that the distribution

of

cysteine was highly conserved. LGP 96 and LGP 107 have one putative transmembrane domain consisting of 24 hydrophobic

amino acids near the COOH-terminus,

and contain a short

cytoplasmic segment composed of 11 amino acid residues at the OH-terminal

end. Similarity

between the transmembrane portions reaches more than 90% if mismatches between chemically similar amino acid residues can be assumed to be identical. The alignment widow was adopted for a similarity calculation as shown in Fig. 4. Similarity between the cytoplasmic tails of them is 69% which is lower than the value obtained from the transmembrane segments. We also compared the COOH-terminal

portions of LGP 96 with those of rat liver lysosomal acid

phosphatase which is anchored to the lysosomal membranes with its CGOH-terminal portion (3). Recently, we isolated and sequenced a cDNA clone corresponding to the entire coding sequence of rat liver lysosomal acid phosphatase (25). This enzyme has one putative transmembrane domain consisting of 27 hydrophobic amino acids near the CGGH-terminus and the M-residue cytoplasmic tail. The CGGH-terminal portions of LGP 96 were compared with those of the acid phosphatase. The results of this comparison are shown in Figs. 4 and 5. The putative transmembrane segments and cytoplasmic tails of these proteins show 75 and 50% similarities, respectively. From the data presented here, we suggest that the transmembrane and cytoplasmic portions of the lysosomal membrane glycoproteins may serve as a molecular signal to target these glycoproteins to lysosomes. Similarity between LGP 96 and h-lamp-2 We compared the deduced amino acid sequence of LGP 96 with that of h-lamp-2 (15), the 120 kDa lysosomal membrane glycoprotein purified from chronic myelogenous leukemia cells. LGP 96 and h-lamp-2 comprise 411 and 408 amino acid residues which include 26 and 28 1118

Vol. 164, No. 3, 1989

BIOCHEMICAL

Name

Amino 400

LGP96 LGP107

AND BIOPHYSICAL

acid

sequence

9 Similarity

410 GLKRH-HTGYEQF . . &iS,i&~TI 400 410 GLKRHHTGYEQF :::.::.::::: GLKHHHAGYEQF 400

LGP96 h-lamp2

RESEARCH COMMUNICATIONS

(9/13)

100%

(12/12)

50%

(6/12)

400

LGP96 APase

410 GLKRHHTGYEQF : :. MQAQPPGYHHV-ADREDHA 410 420

69%

Fi .5. Comparison of amino acid sequencesimilarity of cytoplasmictail of LGP 96 to thoseof

Ehki. ll-lalllD2iilldAPi%.

The cytbplasm;c segmentof LGP 96 (upper) and the counterpartof each protein (lower) are representedin the standardone-letter code, as indicated in Fig. 4.

residues as a signal peptide, respectively. Both proteins contain 8 cysteines and the amino acid is distributed over the molecules in the relatively similar intervals, as mentioned above. LGP 96 and h-lamp-2 have 17- and 16-sites for N-glycosylation, respectively, and these N-glycosylation sites are clustered into two domains separated by the hinge-like structure enriched with proline and threonine. Similarity between the deduced amino acid sequence of LGP 96 and h-lamp-2 reached 65.3%. The putative transmembrane domains in both molecules surprisingly showed a 100% similarity as well as the cytoplasmic tails as shown in Figs. 4 and 5. Thus, LGP 96 and hlamp-2 are probably similar molecules from different species. ACKNOWLEDGMENTS We thank Drs. Y. Ikehara and Y. Misumi for providing the rat liver cDNA library and M. Ohara for helpful comments. This study was supported in part by a grant-in-aid for scientific research from the Ministry of Education, Science and Culture of Japan. REFERENCES 1. Griffiths, G., Hoflack, B., Simons, K., Mellman, I., and Komfeld, S. (1988) Cell 52, 329-341. Komfeld, S. (1987) FASEB (Fed. AM. Sot. Exp. Biol.) J. 1,462-468. i- Himeno, M., Koutoku, H., Ishikawa, T., and Kato, K. (1989) J. B&hem. 105,449-456. 4: Waheed, A., Gottschalk, S., Hille, A., Krentler, C., Pohlmann, R., Braulke, T., Hauser, H., Geuze, H., and von Figura, K.(1988) EMBO J. 7,2351-2358. 5. Sandoval, I.V., Chen, J.W., Yuan, L., and August, J.T. (1989) Arch. B&hem. Biophys. 271, 157-167. 6. Chen, J.W., Pan, W., D,Souza, M.P., and August, J.T. (1985) Arch. B&hem. Biophys. 239,574-586. 7. D,Souza, M.P., and August, J.T. (1986) Arch. Biochem. Biophys. 249,522-532. 8. Kato, K. (1985) in: 3rd International Congress on Cell Biology (Abstracts) Academic Press, Inc., Tokyo, Japan. 9. Himeno, M., Noguchi, Y., Sasaki, H., Tanaka, Y., Furuno, K., Kono, A., Sakaki, Y., and Kate, K. (1989) FEBS L&t. 244, 351-356. 1119

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10. Lewis, V., Green, S.A., Marsh, M., Vinko, P., Helenius, A., and Mellman, I. (1985) J. Cell Biol. 100, 1839-1847. 11. Barriocanal, J.G., Bonifacino, J.S., Yuan, L., and SandovaL1.V. (1986) J. Biol. Chem. 261,16755-16763. 12. Reggio, H., Bainton, D., Harms, R., and Louvard, D. (1984) J. Cell Biol. 99, 1511-1526. 13. Lippincott-Schwartz, J., and Fambrough, D.M. (1986) J. Cell Biol. 102, 1593-1605. 14. Carlsson, S.R., Roth, J., Piller, F., and Fukuda, M. (1988) J. Biol. Chem. 263, 1891118919. 15. Fukuda, M., Viitala, J., Matteson, J., and Carlsson, S.R. (1988) J. Biol. Chem. 263, 18920-18928. 16. Misumi, Y., Tashiro, K., Hattori, M., Sakaki, Y., and Ikehara, Y.(1988) B&hem. J. 249,661-668. 17. Huynh, T., Young, R.A., and Davis, R.W. (1985) in: DNA Cloninig: A Practical Approach 1 (Glover, D.M. ed.) pp. 49-78, IRL Press, Oxford. 18. Vieira, J., and Messing, J. (1982) Gene (Amst.) 19,259-268. 19. Sanger, F., Nicklen, S., and Coulson, A.R.(1977) Proc. Natl. Acad. Sci. USA 74,54635467. 20. Kuhara, S., Matsuo, F., Futamura, S., Fujita, A., Shinohara, T., Takagi, T., and Sakaki, Y.(1984) Nucleic Acids Res. 12, 89-99. 21. Kyte, J., and Doolittle, R.F.(1982) J. Mol. Biol. 157, 105-132. 27. 22. von Heiji, G. (1983) Eur. J. B&hem. 133, 17-21. 23. von Heiji, G. (1985) J. Mol. Biol. 184,99-105. 24. Howe, C.L., Granger, B.L., Hull, M., Green, S.A. Gabel, C.A., Helenius, A., and Melhnan, I. (1988) Proc. Natl. Acad. Sci. USA. 85,7577-7581. 25. Himeno, M., Fujita, H., Noguchi, Y., Kono, A., and Kato, K.(1989) Biochem. Biophys. Res. Commun. 162,1044-1053.

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