©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Strain-specific Presence of Two TGN38 Isoforms and Absence of TGN41 in Mouse (*)

Kazuo Kasai (§) , Senye Takahashi (§) , Kazuo Murakami , Kazuhisa Nakayama (1)(¶)

From the (1)Institute of Applied Biochemistry and the Institute of Biological Sciences and Gene Experiment Center, University of Tsukuba, Tsukuba, Ibaraki 305, Japan

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

TGN38 and TGN41 are isoforms of an integral membrane protein that is predominantly localized to the trans-Golgi network (TGN) in rat cells. They have been proposed to form a heterodimer and to be involved in the budding of exocytic transport vesicles from the TGN. By cDNA cloning and analysis using polymerase chain reaction, we found that there were two TGN38 isoforms in a strain of mouse (ICR), whereas other strains examined (BALB/c, DBA/2, and C57BL/6) had only one TGN38. The major difference between the two isoforms was in the number of characteristic octapeptide repeats. Apart from this, there were several nucleotide substitutions between them. The two isoforms appeared to be derived from two distinct genes but not from one gene via alternative splicing. Furthermore, we failed to show the presence of TGN41 in all the strains examined. This result suggests that TGN38 may function as a monomer or a homodimer in mouse cells.


INTRODUCTION

The trans-Golgi network (TGN)()is a tubuloreticular structure immediately apposed to the trans-most face of the Golgi stack and functions as a sorting machinery for proteins within the secretory pathway. Newly synthesized proteins delivered through the Golgi stack to the TGN are packaged into different types of transport vesicles which are directed to the final destinations, including different domains of the plasma membrane, lysosomes, endosomes, and regulated secretory granules (for review, see Griffiths and Simons, 1986; Burgess and Kelly, 1987; Mellman and Simons, 1992; Rothman and Orci, 1992; Bauerfeind and Huttner, 1993). The molecular mechanism that underlies the sorting on the TGN is not well understood. One well characterized exception is the sorting of lysosomal hydrolases, which bind to mannose 6-phosphate receptor via their mannose 6-phosphate residues, are packaged into clathrin-coated vesicles and are delivered to lysosomes (for review, see Kornfeld and Mellman, 1989; Pearse and Robinson, 1990). Interaction of sorting receptors with cytosolic proteins must be required for the protein sorting on the TGN. For example, mannose 6-phosphate receptor has been shown to interact with HA-I/AP-1 clathrin adaptor via its cytoplasmic tail (Glickman et al., 1989; Le Borgne et al., 1993).

TGN38 is a type I integral membrane protein predominantly localized to the TGN (for review, see Luzio and Banting, 1993; Stanley and Howell, 1993). Its cDNA first isolated from rat liver (Luzio et al., 1990) is deduced to encode a protein with a 17-amino acid signal peptide, a 286-residue luminal domain having characteristic octapeptide repeats and a 57-residue acidic region, a 21-residue transmembrane domain, and a 33-residue cytoplasmic tail. Recently, an isoform of TGN38, designated TGN41, has been identified in rat (Reaves et al., 1992) and suggested to form a heterodimer with TGN38 (Jones et al., 1993; Stanley and Howell, 1993). The only difference between the cDNAs encoding TGN38 and TGN41 is an 8-bp change, including a 5-bp insertion in TGN41, in the region encoding the cytoplasmic tail. The insertion changes the reading frame such that the three COOH-terminal amino acids of TGN38 are not present, and instead an extra 23 amino acids are added in TGN41. Although TGN38/41 is concentrated in the TGN, recent studies have shown that it also cycles between the TGN and the cell surface (Ladinsky and Howell, 1992; Reaves et al., 1993; Bos et al., 1993; Ponnambalam et al., 1994; Chapman and Munro, 1994). Extensive studies have revealed that a tyrosine-containing motif, YQRL, within the cytoplasmic tail serves as a signal for both the TGN localization and the retrieval from the cell surface to the TGN (Bos et al., 1993; Humphrey et al., 1993; Wong and Hong, 1993). Recently, Jones et al.(1993) have shown that the cytoplasmic tail of TGN38/41 binds a cytosolic complex of a 62-kDa protein (p62) and some small GTP-binding proteins including Rab6 and have suggested that the dynamic interaction between the cytosolic complex and TGN38/41 is essential for the budding of exocytic vesicles from the TGN (Stanley and Howell, 1993).

To learn more about the function of TGN38 and TGN41, we first attempted to clone their cDNAs. However, we unexpectedly obtained evidence for the presence of two isoforms of TGN38 in mouse. We therefore began to investigate these TGN38 isoforms in detail.


EXPERIMENTAL PROCEDURES

Animals

All mice used in the present study were purchased from CLEA Japan, Inc. (Tokyo, Japan).

Cloning of Mouse TGN38 cDNAs

Mouse TGN38 cDNA fragments were obtained by reverse transcriptase-polymerase chain reaction (PCR) as follows. Single-stranded cDNA generated from ICR mouse brain total RNA using a first-strand cDNA synthesis kit (Pharmacia-LKB Biotechnology, Uppsala, Sweden) was subjected to PCR amplification using primers synthesized based on the rat TGN38 cDNA sequence (Luzio et al., 1990): 5`-GATAAAGAAGGCCAGGACAAGAC-3` (primer 1; corresponding to nucleotides 201-223) and 5`-GTTTCCACATCTTCAGTAGGCTC-3` (primer 2; complementary to nucleotides 717-739). DNA amplification using a GeneAmp AmpliTaq PCR kit (Perkin-Elmer) was performed in a Perkin-Elmer thermal cycler with 40 cycles of denaturation (94 °C, 2 min), annealing (55 °C, 2 min), and extension (72 °C, 50 s). When electrophoresed on a 5% polyacrylamide gel, two DNA fragments, designated A and B, around the expected size (540 bp) were observed. Both fragments were extracted from the gel, ligated into the SmaI site of pBluescript II (Stratagene, La Jolla, CA), and subjected to sequence analysis. Screening of 3 10 and 2 10 phages from ICR mouse brain and liver cDNA libraries, respectively, in the gt10 vector (Hosaka et al., 1994) using the B fragment as a probe gave rise to 10 and 3 positive clones, respectively. The cDNA inserts subcloned into pBluescript II were subjected to sequence analysis. None of the cDNA inserts thus obtained contained the 5`-end of the coding sequence. To obtain the missing 5`-end of the TGN38 transcripts, we performed extension of ICR mouse brain RNA by the 5`-RACE (rapid amplification of cDNA ends) method (Frohman et al., 1988). Primer 2 was used for the first round of PCR amplification and primer 3, 5`-CCAAACTGTGATCGGTCGCG-3` (complementary to nucleotides 299-318 in Fig. 3), for the second round. Five PCR-derived clones containing the 5`-end of the TGN38 cDNAs (three for TGN38A and two for TGN38B) were sequenced to rule out potential PCR-generated mutations.


Figure 3: Composite nucleotide and deduced amino acid sequences of mouse TGN38A and B. The overall nucleotide and amino acid sequences of mouse TGN38B are shown. The nucleotides and amino acids of TGN38A which differ from those of TGN38B are also shown. Gaps introduced into the alignment are indicated by hyphens.



Northern Blot Analysis

Ten micrograms of total RNAs isolated from tissues of ICR mouse were electrophoresed on an agarose gel and blotted onto a Nylon membrane as described previously (Hatsuzawa et al., 1990). The blot was hybridized with a 308-bp HincII-DraI fragment of the mouse TGN38A cDNA and then washed under conditions as described previously (Hatsuzawa et al., 1990).


RESULTS AND DISCUSSION

To obtain a rat TGN38 cDNA fragment, we carried out reverse transcriptase-PCR of total RNA isolated from rat pheochromocytoma PC12 cells using a set of primers flanking the characteristic octapeptide repeats (see ``Experimental Procedures''). As shown in Fig. 1, an amplified cDNA fragment with an expected size (540 bp) was observed. At the same time, reverse transcriptase-PCR of total RNA from ICR mouse brain was also performed as a control. In this case, however, we unexpectedly found two amplified DNA fragments around 540 bp. We labeled the smaller and larger fragments as A and B, respectively. Two intense bands at 1.2 and 1.5 kilobases were also detected but were confirmed to be nonspecific by sequence analysis (data not shown). As shown in Fig. 2, the nucleotide and deduced amino acid sequences of the A and B fragments were very similar but not identical to each other and were significantly homologous to those of rat TGN38/41 (Luzio et al., 1990). The differences were a 24-bp deletion/insertion and five nucleotide substitutions. Therefore, we tentatively designated the proteins encoded by the A and B fragments as TGN38A and TGN38B, respectively.


Figure 1: Presence of two TGN38 isoforms in mouse. Total RNAs isolated from rat pheochromocytoma PC12 cells and ICR mouse brain were subjected to reverse transcriptase-PCR using a set of primers for rat TGN38. Experimental details are described under ``Experimental Procedures.'' DNA bands corresponding to mouse TGN38A and B are indicated.




Figure 2: Nucleotide and deduced amino acid sequences of PCR-amplified cDNA fragments for mouse TGN38. The nucleotide and amino acid sequences of mouse (m) TGN38A and B are aligned with the pertinent region of rat (r) TGN38 sequences. Nucleotides in the two mouse sequences which differ from each other are shown in italics. Amino acid residues conserved between at least two members are shown in dark boxes. Gaps introduced into the alignment are indicated by hyphens. The sequences of the octapeptide repeats are double underlined.



In an attempt to obtain full-length cDNAs for TGN38A and B, cDNA libraries of ICR mouse brain and liver were screened using the B fragment as the probe. Ten and three positive clones were obtained from the brain and liver libraries, respectively. By restriction endonuclease mapping and partial nucleotide sequencing, four of the brain clones were deduced to code for TGN38A and six for TGN38B, whereas all of the liver clones were for TGN38A. It should be emphasized that no cDNA clone thus obtained encoded a mouse counterpart of the TGN41 isoform (Reaves et al., 1992); this is discussed below. Since none of the clones thus obtained covered the putative translation initiation codon (the longest clones for TGN38A and B started at nucleotides 59 and 30, respectively, in the sequences shown in Fig. 3), we applied the 5`-RACE method (Frohman et al., 1988) using a primer corresponding to the nucleotide sequence common to both types of the cDNAs (see ``Experimental Procedures'').

Fig. 3shows the composite nucleotide sequences of the TGN38A and B cDNAs and their deduced amino acid sequences. TGN38A and B were deduced to consist of 353 and 363 amino acids, respectively (also see Fig. 4). The major differences in the coding sequence between them were deletions/insertions of 6 and 24 bp. The latter was within the characteristic octapeptide repeat region (also see Fig. 2and Fig. 4). Consequently, TGN38A and B have six and seven repeats, respectively. There were also 11 nucleotide substitutions throughout the coding sequence, six of which caused amino acid substitutions. Comparison of the mouse TGN38 sequences with the rat one (Luzio et al., 1990) revealed that the NH-terminal half (amino acids 18-194 in mouse TGN38B) including the octapeptide repeats is not well conserved; the amino acid identity is only 60% between mouse TGN38B and rat TGN38 (Fig. 4). In contrast, the COOH-terminal half (amino acids 195-363 in mouse TGN38B) including the acidic region, the transmembrane domain, and the cytoplasmic domain with the tyrosine-containing TGN localization motif is highly conserved with an amino acid identity of 94%; notably, the sequences containing the transmembrane and the cytoplasmic domains are identical to each other. These data lead to the speculation that the COOH-terminal region plays more important roles in the function and localization of TGN38 than the NH-terminal one does, although the roles of the octapeptide repeat and acidic regions are currently unknown. A part of this speculation is supported by the recent data of Ponnambalam et al.(1994) showing that not only the cytoplasmic domain but also the transmembrane domain contains a TGN localization signal.


Figure 4: Sequence comparison of mouse (m) TGN38 isoforms with rat (r) TGN38. Amino acid residues of mTGN38A and rTGN38 identical with those of mTGN38B are indicated by dots. Gaps introduced into the alignment are indicated by hyphens. Predicted signal peptide and transmembrane domain sequences are shown in dark boxes. The octapeptide repeat sequences are shadowed, and the acidic regions are underlined. The tyrosine-containing motifs are double underlined.



We then examined tissue and cellular distribution of TGN38 transcripts because there has been no report on the TGN38 distribution. Northern blotting of RNAs from mouse tissues and from cultured cell lines of mouse origin revealed that TGN38 mRNAs were expressed ubiquitously (Fig. 5), suggesting that TGN38 plays a fundamental role in most, if not all, types of cells. Three transcripts with different sizes (4.5, 3.3, and 1.8 kilobases) were observed. This size difference can be ascribed to the difference in the length of the noncoding region rather than to one for TGN38A and others for TGN38B or vice versa because cDNA clones with sizes of >1.8 kilobases were obtained for both TGN38A and B (data not shown).


Figure 5: Northern blot analysis. Total RNAs from various ICR mouse tissues and mouse cell lines were probed with a TGN38A cDNA fragment as described under ``Experimental Procedures.''



To determine whether both TGN38 isoforms are expressed ubiquitously, we then performed reverse transcriptase-PCR of RNAs from ICR mouse tissues and mouse cell lines using a set of primers flanking the octapeptide repeats; in this and the following experiments, primers synthesized on the basis of the mouse TGN38 sequence were used in place of those for the rat TGN38 sequence used in the above experiments because there were some mismatched nucleotides between sequences of the rat primers and the mouse TGN38 cDNA. As shown in Fig. 6A, DNA fragments for both TGN38A and B were observed in all examined tissues, indicating widespread expression of both isoforms; two intense bands at 420 and 500 bp were also detected but were confirmed to be nonspecific (data not shown). However, we unexpectedly found only one fragment corresponding to the position of TGN38A from RNAs from cultured cell lines of mouse origin, NIH3T3, AtT-20, and L929. One possibility to which this observation is ascribed is that the presence of the two TGN38 isoforms is a strain-specific event because the cell lines used originate from different mouse strains. To address this possibility, we performed reverse transcriptase-PCR of liver RNAs from different mouse strains. As shown in Fig. 6B, only TGN38A was present in the BALB/c, C57BL/6, and DBA/2 strains, whereas both isoforms were in the ICR strain. We also performed PCR of genomic DNAs from BALB/c and ICR strains to determine whether the absence of the TGN38B transcript is for lack of the TGN38B gene itself or for lack of its transcription. As shown in Fig. 6C, although two bands for TGN38A and B were detected in the ICR strain, one band for TGN38A was in the BALB/c strain. Sequence analysis of the amplified DNA fragments revealed not only that the two genomic fragments of ICR coded for TGN38A and B, and the one of BALB/c for TGN38A, but also that there were no intervening sequence within the amplified region including the octapeptide repeats (data not shown). These data indicate that the presence of TGN38B is a strain-specific event. Furthermore, taken together with the fact that substitutions between TGN38A and B disperse throughout the nucleotide sequence (see Fig. 3), these data indicate that the two isoforms in ICR mouse derive from two distinct genes but not from the same gene via alternative splicing. To understand the origin of the two TGN38 genes, a phylogenetically based survey of laboratory and wild mice will be required.


Figure 6: Strain-specific presence of TGN38B. Panel A, single-stranded cDNAs synthesized from RNAs of ICR mouse tissues and mouse cell lines were subjected to PCR using a set of primers: 5`-GTCAACCCCAAACTGGAAAAGTC-3` (primer 4; corresponding to nucleotides 387-409 in Fig. 3) and 5`-TTTTTCAGTTGGCTGGACTTTGTCA-3` (primer 5; complementary to nucleotides 626-650 in Fig. 3). DNA amplification using a GeneAmp AmpliTaq PCR kit was performed in a thermal cycler with 35 cycles of denaturation (94 °C, 2 min), annealing (65 °C, 2 min), and extension (72 °C, 45 s). The PCR products were electrophoresed on a 5% polyacrylamide gel. Panel B, single-stranded cDNAs synthesized from liver RNAs of BALB/c, C57BL/6, DBA, and ICR strains were subjected to PCR under conditions described above. Panel C, genomic DNAs of BALB/c and ICR strains were subjected to PCR under conditions described above.



As described above, we could not obtain a cDNA for TGN41; amino acid sequences deduced from all 13 cDNA clones of mouse brain and liver possessed a TGN38-type cytoplasmic tail sequence. This led to the possibility that TGN41 does not exist in mice. To address this possibility, cDNAs synthesized from RNAs of various mouse tissues and cell lines, and mouse genomic DNAs were subjected to PCR analysis using primers flanking the sequence where TGN38 and TGN41 should differ from each other (Fig. 7A). In rat, there is an 8-bp change including a 5-bp insertion in TGN41. Therefore, using these primers, the TGN38 sequence should give a 76-bp fragment, whereas the TGN41 sequence, if present, should give an 81-bp fragment. As shown in Fig. 7B, only one fragment around 76 bp was amplified from RNAs of ICR and BALB/c mouse tissues and mouse cell lines, and from genomic DNAs of ICR and BALB/c mice. Identical results were obtained using liver RNAs of C57BL/6 and DBA/2 mice (data not shown). A rare possibility that two DNA fragments with a difference of 5 bp in length could migrate with the same mobility in the gel can be excluded by the following three criteria. (i) By HindIII digestion, the TGN38 PCR product should be cut into two fragments of 34 and 42 bp, whereas the TGN41 product, if present, should be cut into 39- and 42-bp fragments (see Fig. 7A). As shown in Fig. 7C, the endonuclease digestion gave rise to 34- and 42-bp fragments but not a 39-bp one. (ii) The sequence alteration and 5-bp insertion in rat TGN41 cDNA introduce a HinfI restriction site that is not found in rat and mouse TGN38 cDNAs (see Fig. 7A). However, all of the PCR products examined remained intact after the HinfI digestion (data not shown). (iii) Finally, we subcloned the PCR products from liver RNAs and from genomic DNAs of both ICR and BALB/c mice, determined the sequences of some independent clones in each case, and found that all of the clones had a TGN38-type sequence. It is also of note that the PCR products from the genomic DNAs did not contain a putative intervening sequence; this excludes the possibility that a TGN41-type transcript could be generated via alternative splicing. Our data thus suggest the absence of a TGN41 transcript and its gene in mouse, although further studies will be required for us to draw a firm conclusion.


Figure 7: Absence of TGN41. Panel A, nucleotide and amino acid sequence of the pertinent region of mouse (m) mTGN38A and mTGN38B and rat (r) rTGN38 and rTGN41. Primers used for PCR analysis are also shown. Gaps introduced into the alignment are indicated by hyphens, and nucleotides that differ between TGN38 and TGN41 are shadowed. Recognition sequences for HindIII and HinfI are underlined and double underlined, respectively. Panel B, single-stranded cDNAs synthesized from RNAs of ICR and BALB/c mouse tissues and mouse cell lines, and genomic DNAs of ICR, and BALB/c mice were subjected to PCR using primers 6 and 7. DNA amplification using a GeneAmp AmpliTaq PCR kit was performed in a thermal cycler with 35 cycles of denaturation (94 °C, 1 min), annealing (68 °C, 2 min), and extension (72 °C, 45 s). The PCR products were electrophoresed on a 15% polyacrylamide gel. Panel C, the PCR products as above were digested with (+) or without (-) HindIII and were electrophoresed on a 20% polyacrylamide gel.



Reaves et al.(1992) have cloned a cDNA encoding TGN41 from a rat liver library and shown the presence of its transcript in rat epididymis by reverse transcriptase-PCR analysis. On the basis of experimental evidence obtained using a rat liver Golgi fraction, Howell and colleagues have proposed that TGN38 and TGN41 form a heterodimer and are involved in the budding of exocytic transport vesicles from the TGN by forming a complex with cytosolic proteins (Jones et al. 1993; Stanley and Howell, 1993). In the present study, however, we failed to show the presence of TGN41 in mouse. A possible explanation for the present observations is that TGN38 functions as a homodimer or a monomer in mouse cells. Since the sequence common to both TGN38 and TGN41 is thought to have the signal that is essential for their TGN localization as discussed by Reaves et al. (1992), TGN41 may be dispensable. To address whether the presence of TGN41 is a rat-specific event, studies are under way to examine whether other species have TGN41.


FOOTNOTES

*
This work was supported in part by grants from the Ministry of Education, Science and Culture of Japan, the Special Research Project on Circulation Biosystem in University of Tsukuba, the Saneyoshi Scholarship foundation, the Nissan Science Foundation, and Sankyo Co., Ltd. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBank/EMBL Data Bank with accession number(s) D50031 and D50032.

§
The first two authors contributed equally to this work.

To whom correspondence should be addressed: Institute of Biological Sciences, University of Tsukuba, Tsukuba, Ibaraki 305, Japan. Tel.: 81-298-53-6356; Fax: 81-298-53-6006.

The abbreviations used are: TGN, trans-Golgi network; bp, base pair(s); PCR, polymerase chain reaction; RACE, rapid amplification of cDNA ends.


ACKNOWLEDGEMENTS

We thank Drs. George Banting and Yukio Ikehara for critical reading of the manuscript and Dr. Kaichiro Yanagisawa for encouragement.


REFERENCES
  1. Bauerfeind, R., and Huttner, W. B.(1993) Curr. Opin. Cell Biol.5, 628-635 [Medline] [Order article via Infotrieve]
  2. Bos, K., Wraight, C., and Stanley, K. K.(1993) EMBO J.12, 2219-2228 [Abstract]
  3. Burgess, T. L., and Kelly, R. B.(1987) Annu. Rev. Cell Biol.3, 243-293 [CrossRef]
  4. Chapman, R. E., and Munro, S.(1994) EMBO J.13, 2305-2312 [Abstract]
  5. Frohman, M. A., Dush, M. K., and Martin, G. R.(1988) Proc. Natl. Acad. Sci. U. S. A.85, 8998-9002 [Abstract]
  6. Glickman, J. N., Conibear, E., and Pearse, B. M. F.(1989) EMBO J.8, 1041-1047 [Abstract]
  7. Griffiths, G., and Simons, K.(1986) Science234, 438-443 [Medline] [Order article via Infotrieve]
  8. Hatsuzawa, K., Hosaka, M., Nakagawa, T., Nagase, M., Shoda, A., Murakami, K., and Nakayama, K.(1990) J. Biol. Chem.265, 22075-22079 [Abstract/Free Full Text]
  9. Hosaka, M., Murakami, K., and Nakayama, K.(1994) Biomed. Res.15,383-390
  10. Humphrey, J. S., Peters, P. J., Yuan, L. C., and Bonifacino, J. S. (1993) J. Cell Biol.120, 1123-1135 [Abstract]
  11. Jones, S. M., Crosby, J. R., Salamero, J., and Howell, K. E.(1993) J. Cell Biol.122, 775-788 [Abstract]
  12. Kornfeld, S., and Mellman, I.(1989) Annu. Rev. Cell Biol.5, 483-525 [CrossRef]
  13. Ladinsky, M. S., and Howell, K. E.(1992) Eur. J. Cell Biol.59, 92-105 [Medline] [Order article via Infotrieve]
  14. Le Borgne, R., Schmidt, A., Mauxion, F., Griffiths, G., and Hoflack, B. (1993) J. Biol. Chem.268, 22552-22556 [Abstract/Free Full Text]
  15. Luzio, J. P., and Banting, G.(1993) Trends Biochem. Sci.18, 395-398 [CrossRef][Medline] [Order article via Infotrieve]
  16. Luzio, J. P., Brake, B., Banting, G., Howell, K. E., Braghetta, P., and Stanley, K. K.(1990) Biochem. J.270, 97-102 [Medline] [Order article via Infotrieve]
  17. Mellman, I., and Simons, K.(1992) Cell68, 829-840 [Medline] [Order article via Infotrieve]
  18. Pearse, B. M. F., and Robinson, M. S.(1990) Annu. Rev. Cell Biol.6, 151-171 [CrossRef]
  19. Ponnambalam, S., Rabouille, C., Luzio, J. P., Nilsson, T., and Warren, G.(1994) J. Cell Biol.125, 253-268 [Abstract]
  20. Reaves, B., Wilde, A., and Banting, G.(1992) Biochem. J.283, 313-316 [Medline] [Order article via Infotrieve]
  21. Reaves, B., Horn, M., and Banting, G.(1993) Mol. Biol. Cell4, 93-105 [Abstract]
  22. Rothman, J. E., and Orci, L.(1992) Nature355, 409-415 [CrossRef][Medline] [Order article via Infotrieve]
  23. Stanley, K. K., and Howell, K. E.(1993) Trends Cell Biol.3, 252-255 [Medline] [Order article via Infotrieve]
  24. Wong, S. H., and Hong, W.(1993) J. Biol. Chem.268, 22853-22862 [Abstract/Free Full Text]

©1995 by The American Society for Biochemistry and Molecular Biology, Inc.