©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
Molecular Characterization of trans-Golgi p230
A HUMAN PERIPHERAL MEMBRANE PROTEIN ENCODED BY A GENE ON CHROMOSOME 6p12-22 CONTAINS EXTENSIVE COILED-COIL alpha-HELICAL DOMAINS AND A GRANIN MOTIF (*)

(Received for publication, October 3, 1995; and in revised form, December 28, 1995)

Rebecca Erlich Paul A. Gleeson Paul Campbell (1)(§) Erin Dietzsch (2) Ban-Hock Toh (¶)

From the  (1)Department of Pathology and Immunology, Monash University Medical School, Melbourne, Victoria 3181, Australia, the Department of Clinical Immunology, Flinders Medical Center, Bedford Park, South Australia, and the (2)Department of Medical Biochemistry, University of Cape Town Medical School, Observatory, Cape Town 7925, South Africa

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Using autoantibodies from a Sjögren's syndrome patient, we have previously identified a 230-kDa peripheral membrane protein associated with the cytosolic face of the trans-Golgi (Kooy, J., Toh, B. H., Pettitt, J. M., Erlich, R. and Gleeson, P. A.(1992) J. Biol. Chem. 267, 20255-20263). Here we report the molecular cloning and sequence analysis of human p230 and the localization of its gene to chromosome 6p12-22. Partial cDNA clones, isolated from a HeLa cell cDNA library using autoantibodies, were used to obtain additional cDNAs, which together span 7695 base pairs (bp). The p230 mRNA is 7.7 kilobases. Two alternatively spliced mRNAs for p230 were detected. These differed by 21- and 63-bp insertions in the 3`-sequence, resulting in differences in amino acid sequence at the carboxyl terminus. The predicted 261-kDa protein is highly hydrophilic with 17-20% homology with many proteins containing coiled-coil domains. Apart from two proline-rich regions (amino acids 1-117 and 239-270), p230 contains a very high frequency of heptad repeats, characteristic of alpha-helices that form dimeric coiled-coil structures. p230 also includes the sequence ESLALEELEL (amino acids 538-546), a motif found in the granin family of acidic proteins present in secretory granules of neuroendocrine cells. This is the first report of a cytosolic Golgi protein containing a granin motif. The structural characteristics of p230 indicate that it may play a role in vesicular transport from the trans-Golgi.


INTRODUCTION

The Golgi apparatus is a highly complex and dynamic organelle organized into three functionally distinct regions: the cis, medial, and trans cisternae of the Golgi stack and two tubulovesicular networks, namely the cis-Golgi network and the trans-Golgi network(1, 2) . Transport of newly synthesized proteins from the endoplasmic reticulum to Golgi cisternae, between adjacent cisternae, and from the cisternae to various destinations is mediated by vesicles shuttling between donor and recipient compartments(3) . Numerous structural and regulatory proteins have been implicated in the budding, docking, and fusion of vesicles(3, 4, 5) .

Soluble proteins involved in budding of vesicles include COPI and COPII coat proteins and the small GTP binding protein ARF-1. A N-ethylmaleimide-sensitive fusion protein, soluble N-ethylmaleimide-sensitive fusion protein attachment proteins (SNAPs) (^1)and Rabs are involved in either vesicle docking or membrane fusion(3, 6, 7, 8) . SNAP receptors (SNAREs), membrane proteins that form oligomeric complexes with SNAPs and N-ethylmaleimide-sensitive fusion proteins, are considered to promote fusion of vesicles with target membranes after specific docking mediated by SNAREs on vesicle and target membranes (3) . However, many facets of the transport process remain unresolved. For example, the protein coat structures that mediate forward vesicle transport from the Golgi apparatus have not been fully characterized.

Additional peripheral membrane Golgi proteins have also been implicated in vesicular transport, for example three high molecular weight proteins in cis to medial Golgi transport(9) . One of these, p115, contains coiled-coil domains and is related to Uso1p required for transport from endoplasmic reticulum to Golgi complex in Saccharomyces cerevisiae(10) . Two peripheral membrane proteins have been implicated in budding of vesicles from the trans-Golgi network, namely p200, which associates with coated vesicles arising from the trans-Golgi network(11) , and p62, which forms a complex with TGN38/41 and Rab6(12) .

Human autoantibodies are valuable reagents for identification of novel intracellular proteins. Using anti-Golgi autoantibodies from a patient with Sjögren's syndrome, we have previously identified a brefeldin A-sensitive peripheral membrane protein of 230 kDa (p230) localized to the cytosolic face of trans-Golgi(13) . Other novel Golgi proteins have also been identified using autoantibodies, including golgins-95 and -160(14) , a protein of 370 kDa(15, 16) , which appears to be identical to giantin(17) , and a cis-Golgi network p210 protein(18) . For those Golgi proteins where sequences are known, a common structural feature is a high content of predicted coiled-coil domains. Here we have cloned and sequenced p230. The predicted protein consists of alpha-helical coiled-coil domains with abundant heptad repeats and contains a granin motif shared with proteins found in secretory vesicles. We propose that p230 has a role in membrane transport of proteins from the Golgi apparatus.


EXPERIMENTAL PROCEDURES

Autoimmune Sera

Autoimmune sera were obtained from Gribbles Pathology (Melbourne, Australia). Sera were treated at 56 °C for 30 min to inactivate complement, and aliquots in 0.02% sodium azide were stored at -70 °C.

Immunofluorescence

Indirect immunofluorescence using human Hep2 cells (Kallstead) was performed as described previously(13) .

Immunoblotting

Immunoblotting of HeLa cell extracts and recombinant p230 bacterial fusion protein was performed as described previously(13) .

Isolation and Sequencing of cDNA Clones

10^5 recombinants of a gt11 HeLa cell cDNA library (Clontech HL1022) were immunoscreened with autoimmune serum from a Sjögren's syndrome patient diluted 1:1000 in phosphate-buffered saline (PBS) as described previously(13) . Positive plaques, detected with I-labeled protein A, were rescreened and plaque-purified. These cDNA clones were subcloned into pBluescribe M13 (Stratagene) for sequencing. Additional cDNA clones were obtained by subsequently screening a ZAP Hepatoma cDNA library (Stratagene 935202) with P-labeled cDNA probes. Positive plaques were purified, and plasmids were rescued according to the manufacturer's instructions. The P-labeled cDNA probes were derived initially from the gt11 clones and subsequently from ZAP clones. In addition, a P-labeled 45-mer based on sequence from a ZAP cDNA clone was constructed from partially overlapping 30-mers, 5`-GAGCTAATCAACATTAGTAGTAGTAAAACT-3` and 5`-AGAAAGAATGGCATTAGTTTTACTACTACT-3` by a method described by Alderuccio et al.(19) . Finally, a random-primed HeLa cell cDNA library in pUEX (20) was screened with a P-labeled 240-bp fragment obtained by PCR using a ZAP clone as template. Nucleotide sequence of cDNA clones clone was determined by the dideoxy method, using a deaza reagent sequencing kit (Promega) in conjuction with T7 DNA polymerase (Pharmacia Biotech Inc.) or by automated sequencing using a Prism DyeDeoxy terminator cycle kit and a 373A DNA sequencer (Applied Biosystems).

Elution of Antibody from Plaques

To induce the expression of beta-galactosidase fusion protein, nitrocellulose filters impregnated with 10 mM isopropyl-1-thio-beta-D-galactopyranoside were overlaid on agar plates containing gt11 plaques for 3 h at 42 °C. The filters were then washed in PBS, blocked in 3% casein, and incubated with autoimmune serum, diluted 1:1000 in PBS, for 1 h at room temperature. After washing in PBS, the bound antibodies were eluted from the filters by gently rocking in 3 M KSCN. The eluate was dialyzed overnight in PBS and concentrated approximately 20-fold using Centricon filters (Amicon).

Reverse Transcriptase Polymerase Chain Reaction

Total RNA was isolated from HeLa cells by guanidium thiocyanate extraction followed by ultracentrifugation in cesium chloride(21) . After centrifugation, the RNA pellets were resuspended in formamide for storage and precipitated in four volumes of ethanol before use(22) . HeLa cell RNA (2 µg) was heated to 70 °C for 2 min, and cDNA was synthesized using 5 µg/ml oligo(dT) (Promega) and 100 units of murine superscript reverse transriptase (Life Technologies, Inc.) in 50 mM KCl, 10 mM Tris-HCl, pH 8.3, 4 mM MgCl(2), 0.01% gelatin, 1 mM of each of the four deoxynucleotide triphosphates (Pharmacia), and 20-40 units of RNAsin (Promega) at 37 °C for 2 h. Reactions were also carried out without reverse transcriptase or without RNA. Half of the volume of these reactions was used as templates for PCR. Oligonucleotide primers were used at 0.2 pmol/µl in the presence of 10 mM Tris-Cl, pH 8.3, 50 mM KCl, 1.5 mM MgCl(2), 0.01% gelatin, 0.2 mM of each of the four deoxynucleotide triphosphates, 2.5 units of Taq polymerase (Life Technologies, Inc.), and 0.016 units of cloned Pfu DNA polymerase (Stratagene). The oligonucleotide primers (produced by Department of Microbiology, Monash University or by Bresatec, Adelaide, Australia) were as follows: P1 (5`-GCTCTAGATCAAGGAGGAGACGGCGA-3`), P2 (5`-CCTCAGTAAGACTTTCTA-3`), P3 (5`-CCCAAGTCTATGAGTCCA-3`), P4 (5`-GCGTCGACCACTGCCAACAATCACAG-3`), and P5 (5`-TTGGTCCAACCCAAATTG-3`). Cycle conditions for P1/P2 and P4/P5 were as follows: 1 cycle of 5 min at 95 °C, 1.5 min at 55 °C, and 3 min at 72 °C and 29 cycles of 1.5 min at 95 °C, 1.5 min at 55 °C, and 3 min at 72 °C. Cycle conditions for P3/P4 were as above except that the annealing temperature was 60 °C.

Southern Blot Analysis

PCR products were separated by agarose gel electrophoresis and then transfered to nylon membranes (Hybond-N, Amersham Corp.) in 0.4 M NaOH for 4 h. Membranes were then incubated with prehybridization buffer (0.9 M NaCl, 90 mM Na(3)C(6)H(5)O(7).2H(2)O, 10 mM EDTA, pH 7.0, 7% SDS, and 0.5% skim milk powder) for 30 min at 65 °C and hybridized overnight at 65 °C with P-labeled DNA (specific activity, >5 times 10^8 cpm/µg) in prehybridization buffer. Membranes were washed at 65 °C in 0.15 M NaCl, 0.015 M Na(3)C(6)H(5)O(7)bullet2H(2)O, pH 7.0, containing 0.1% SDS and exposed to x-ray film (Fuji, Ashigara, Japan).

Northern Blot Analysis

- Total RNA was prepared from HeLa cells as described above and enriched for poly(A) RNA by oligo(dT)-cellulose chromatography using a poly(A) tract mRNA isolation system (Promega). Poly(A) RNA was separated by electrophoresis on 1% agarose formaldehyde gel and transferred to nylon membranes (Hybond-N, Amersham Corp.) in 0.05 M NaOH for 2 h. After transfer, lanes containing RNA markers were cut into strips and stained in methylene blue(21) . The remaining membrane was incubated with P-labeled DNA probes as described for Southern analysis. The membranes were stripped for reprobing by the addition of 0.5% SDS at 100 °C followed by cooling to room temperature. The removal of the P-labeled probe was confirmed by autoradiography.

Sequence Analysis

Analyses were carried out using the MacVector program (International Biotechnologies, Inc) or using on-line software provided by the Australian National Genome Information Service including the FASTA program,(23) , the pattern library search program of Smith and Smith(24) , and various programs contained in the University of Wisconsin GCG package(25) . Coiled-coil structures were predicted using the method of Lupas et al.(26) using the STRIPE program for the Macintosh. (^2)

Chromosome in Situ Hybridization

Human chromosome metaphase spreads were obtained from PHA-stimulated peripheral blood lymphocyte cultures synchronized with fluorodeoxyuridine (28) according to standard cytogenetic procedures. The method of in situ hybridization was modified from Harper and Saunders (29) and Choo et al.(30) . The g5 clone, labeled by random priming using [^3H]dATP, [^3H]dCTP, and [^3H]dTTP to a specific activity of 2.04 times 10^8 cpm/µg DNA was added to the hybridization solution and hybridized to chromosome preparations overnight at 40 °C. After washes in 0.15 M NaCl, 0.015 M Na(3)C(6)H(5)0(7)bullet2H(2)0, pH 7.0, at 42 °C, slides were dipped in EM-1 hypercoat nuclear emulsion (Amersham Corp.) and exposed for 8-33 days at 4 °C. Autoradiographs were developed, and chromosomes were G-banded in 2-5% Gurr's improved R66 Giemsa stain (Merck, Kilsyth, Australia) in 0.15 M Na(2)HPO(4), pH 11, for 8 min(31) . The distribution of silver grains over chromosomes was analyzed.


RESULTS

Isolation and Characterization of cDNA Clones

We have identified two autoimmune sera that show strong staining of the Golgi apparatus by immunofluorescence (Fig. 1) and that immunoblot a 230-kDa protein in HeLa cell extracts (Fig. 2). One of these sera (autoimmune serum 1), from a patient with Sjögren's syndrome(13) , has high titre anti-Golgi autoantibodies by immunofluorescence (1:100,000) as well as lower titre (1:20,000) antinuclear antibodies. Immunoscreening a HeLa cell gt11 cDNA expression library with diluted Sjögren's syndrome autoimmune serum yielded four clones designated g2, g5, g7, and g12 (Fig. 3). Autoantibodies eluted from nitrocellulose lifts of purified phage from each of the four clones showed immunofluorescence staining of the Golgi apparatus, whereas elution of bound antibodies from an irrelevant clone showed no staining (Fig. 1). Furthermore, the eluted autoantibodies showed no nuclear staining. These results indicate that all four clones encode Golgi-associated proteins. Clone g5 was used to generate a bacterial fusion protein, using the expression vector pGEX(13) . The two autoimmune sera specifically reacted with the 51-kDa glutathionine S-transferase fusion protein by immunoblotting (Fig. 2; see also (13) ). As reported previously(13) , affinity-purified Sjögren's syndrome autoantibody eluted from the 51-kDa recombinant fusion protein not only reacted with the Golgi apparatus in HeLa cells by immunofluorescence but also immunoprecipitated and immunoblotted a 230-kDa protein; furthermore, a rabbit antibody raised to the 51-kDa fusion protein gave specific immunofluorescence of the Golgi apparatus and immunoblotted and immunoprecipitated the 230-kDa protein. These results confirm that clone g5 encodes a polypeptide derived from p230.


Figure 1: Intracellular localization of autoantigens by indirect immunofluorescence. Human Hep2 cells were stained by indirect immunofluoresence with autoimmune serum 1 (a) and autoimmune serum 2 (b) or with autoantibodies from serum 1 eluted from clone g5 (c) or from an irrelevant gt11 clone (d). Magnifications: a, times 1000; b, times 400; c, times 800; d, times 500.




Figure 2: Immunoblot analysis of cell extracts and bacterial fusion protein with autoimmune serum 2. HeLa cell proteins (A) and total proteins from isopropyl-1-thio-beta-D-galactopyranoside-treated E. coli DH1 cells transformed with recombinant pGEX-clone g5 autoantigen (B) were separated under reducing conditions on a 5 or 7.5% polyacrylamide gel, respectively, and transferred to nitrocellulose membranes. Membranes were incubated with autoimmune serum 2 (AIS) or normal human serum (NHS) followed by peroxidase-conjugated anti-human immunoglobulin. Bound immunoglobulin was detected by Enhanced Chemiluminesence.




Figure 3: Map of p230 cDNA clones. Clones g2, g5, g7, and g12 were isolated from a gt11 HeLa cell cDNA library by immunoscreening with autoimmune serum 1. A ZAP hepatoma cDNA library was screened with g5 yielding clones z2 and z6; screening the same library with z6 resulted in clones z7a and z8a, which also showed positive hybridization with g5. The ZAP Hepatoma cDNA library was also screened with a 300-bp fragment of g7 resulting in z7, z8, and z16, while a 45-bp segment of z6 identified z9. Finally, a PCR-generated fragment of the 5` region of z9 was used to screen a pUEX HeLa cell cDNA plasmid library, identifying clone px1.



The four clones (g2, g5, g7, and g12) showed an overlapping nucleotide sequence spanning approximately 2.0 kb (Fig. 3). To obtain additional sequence, we recreened a ZAP cDNA hepatoma library using clone g5 as probe. Two further clones, designated z2 and z6, were isolated (Fig. 3). Further screening of the same library with z6 resulted in clones z7a and z8a. These latter clones also gave positive hybridization signals with clone g5, and DNA sequencing confirmed their overlapping sequence. Screening the ZAP hepatoma library with a 300-bp HincII fragment of g7 identified clones z7, z8, and z16, and screening with a 45-bp segment of z6 identified z9 (Fig. 3). The 5`-sequence was obtained from clone px1, isolated by screening a randomly primed HeLa cell plasmid cDNA library with a PCR-generated 240-bp fragment of the 5`-region of z9. Together, all of the clones (Fig. 3) comprise 7.7 kb of cDNA, as determined by nucleotide sequencing. Three of the cDNA clones, namely px1, z9, and z16, collectively span the entire 7.7-kb cDNA.

Northern analysis showed that the three clones, px1, z9, and z16 all hybridized with a similar sized transcript from HeLa cell poly(A) RNA of about 7.7 kb (Fig. 4a). To confirm that the overlapping clones shown in Fig. 3were derived from the same transcript, reverse transcriptase PCR was carried out using total RNA isolated from HeLa cells and oligonucleotide primers as indicated in Fig. 4b. A 3.2-kb product was obtained using primers P1 and P2, and a 4.7-kb product was generated using primers P3 and P4; the sizes of these PCR products were in accordance with that expected from the nucleotide sequence of the clones. The identity of the PCR products was confirmed by Southern blot analysis using internal probes. The P1/P2 product was probed with a 1.3-kb fragment from clone px1 and the P3/P4 product with the above mentioned 45-mer (Fig. 4b). Taken together, our data imply that we have isolated a full-length cDNA encoding p230.


Figure 4: A, Northern blot analysis. HeLa cell poly (A) RNA was size fractionated by formaldehyde gel electrophoresis, transferred to Hybond N membranes and hybridized with P-labeled cDNA from clone px1. After washing the membrane and visualizing the signal by autoradiography, the P-labeled DNA was stripped from the membrane, as described under ``Experimental Procedures,'' and the membrane reprobed with P-labeled DNA from clone z9. A separate membrane was probed with P-labeled DNA from clone z16. B, reverse transcriptase PCR and Southern blot analysis. The three clones, px1, z9, and z16, which span the full length of the p230 cDNA overlap as indicated. To determine if the three clones are derived from the same mRNA, total RNA from HeLa cells was reverse transcribed, and the cDNA amplified using oligonucleotide primers P1 and P2 or P3 and P4. The expected sizes of the products are indicated. Incubations were also carried out in the absence of either RNA or reverse transcriptase. PCR products were analyzed by Southern blotting using independent internal cDNA probes, as described under ``Experimental Procedures.'' The generation of PCR products of the expected size confirms the relationship of these overlapping clones.



The clones shown in Fig. 3gave identical sequences in the overlap regions. The nucleotide sequence reported here was verified by either sequence of both strands of a cDNA clone, identical sequences from at least one other independent clone, or from reverse transcriptase PCR products. However, two small differences were detected in the three 3` clones. These differences comprised a 21-bp stretch (nucleotide 6592-6612) present in clones z7 and z8, but absent in z16 and a 63-bp stretch (nucleotide 6950-7012) present in z7 but absent from clones z16 and z8 (Fig. 5). The absence of the 21 nucleotides does not disrupt the open reading frame but results in the loss of the amino acids VTIMELQ, while the absence of the 63 nucleotides results in insertion of an alternative stop codon and the amino acid sequence of p230 ending in SWLRSSS rather than FTSPRSGIF. Reverse transcriptase PCR using oligonucleotide primers P4 and P5 resulted in two products of 1.5 and 1.6 kb, sizes consistent with the 63- and 21-bp insertion and deletion (Fig. 6). There is a unique HincII site in the 63-bp insertion, and as expected, the 1.6-kb PCR product was susceptible to HincII digestion, giving the expected fragments of 0.98 and 0.64 kb. These results indicate the presence of alternatively spliced mRNAs derived from the same gene. Whether these different mRNAs represent regulated splicing events or products from inaccurate splicing is not known.


Figure 5: Identification of two p230 mRNA species. A, nucleotides 6592-6612 are present () in clones z7 and z8, but absent (box) from clone z16. Nucleotides 6950-7012 are present () in clone z7, but absent (box) from clones z16 and z8. The sequence from 6950-7012 () contains an in-frame TGA stop codon and a HincII restriction site. P5 (18-mer) and P4 (24-mer) are oligonucleotides used for reverse transcriptase PCR to assess the presence of more than one p230 transcript. The expected sizes of the PCR products are indicated. B, total RNA from HeLa cells was reverse transcribed using oligo(dT), and the resulting cDNA were amplified using primers P4 and P5. The PCR product was divided into two, with one-half digested with HincII. Samples were separated by agarose gel electrophoresis and visualized by ethidium bromide staining. Incubations were also carried out in the absence of either RNA or reverse transcriptase, as indicated.





Figure 6: cDNA and predicted amino acid sequence of p230. Nucleotide sequence of p230 is shown, together with the predicted sequence of the encoded polypeptide. Proline-rich regions of the predicted polypeptide (see text; domains 1 and 3) are shaded and the granin signature is boxed.



Sequence Analysis

The nucleotide sequence of p230 cDNA comprises 7695 bp and contains an open reading frame of 6690 bp (Fig. 6). The open reading frame spans an in-frame ATG initiation codon at nucleotide position 286 and a TGA termination codon at position 6976. A second potential ATG initiation codon is located at nucleotide 388. Both initiation codons have a purine residue 3 bases 5` upstream of the ATG, representing favorable translation initiation sites(32) . Based on the linear scanning model of translation initiation(33) , the first AUG initiation codon would be expected to be the dominant signal. The TGA codon is followed by a polyadenylation signal AATAAA (34) at position 7660 and a poly(A) tail from position 7679 to 7694.

The open reading frame encodes a putative polypeptide of 2230 amino acids with a predicted molecular weight of 261,126 Da and a pI of 5.16. The amino acid composition of this putative polypeptide is rich in glutamic acid (16.6%), lysine (12.7%), leucine (12.5%), and glutamine (10.1%) residues. Comparison with the GenBank(TM) data base reveals regions of p230, which are identical or nearly identical to 15 previously isolated partial cDNA clones of no known function. A list of these loci together with the corresponding regions of identity in p230 is given in Table 1.



Comparison of the translated amino acid sequence of p230 with the translated GenBank(TM) data base reveals significant homology (17-27% identity) with many proteins known or predicted to encode coiled-coil domains. These include various conventional and nonconventional myosins, tropomyosins, cytokeratins, vimentin, neurofilaments, laminin, hemeolytic streptococcal M proteins, dystrophin, the Golgi proteins golgin-95, golgin-160, and giantin, the Uso1 protein of S. cerevisiae implicated in endoplasmic reticulum to Golgi transport and its mammalian homologue p115 or TAP, and the early endosome-associated protein EEA-1(35) . In addition, this homology was shared with other structural and motor proteins of the cytoskeleton including human cytoplasmic linker protein-170, kinetocore protein CENP-E, lamins, dynein, and kinesins. In general, this pattern of identity was scattered throughout the length of p230, although similar comparisons with translated GenBank(TM) sequences using a series of overlapping segments of p230 revealed that such homology was less pronounced in the amino-terminal 130 amino acids of p230.

There are two proline-rich domains at the amino terminus of the putative polypeptide; amino acids 1-117 contains 6.8% proline residues, while the segment from amino acids 239-270 contains 18.8% proline residues (Fig. 6), suggesting a compact structure for these domains. The remainder of the protein has scant proline residues, most of which are clustered at the extreme carboxyl terminus. Thus, p230 can be divided into four putative domains on the basis of its proline distribution, i.e. domain 1 (amino acids 1-117), domain 2 (amino acids 118-238), domain 3 (amino acids 239-270), and domain 4 (amino acids 271-2230).

These analyses are consistent with the two proline-rich domains having a high probability of globular structures and an elongated structure for the intervening domains 2 and 4(36) . These features are supported by results of secondary structure predictions (Fig. 7), which suggest a predominantly alpha-helical structure for p230 with the exception of the proline-rich domains 1 and 3. Analyses of hydrophilicity (Fig. 7) suggest a predominantly hydrophilic structure, with no evidence for a hydrophobic transmembrane domain, consistent with biochemical data reported by Kooy et al.(13) . Charge plots (not shown) show no evidence for discrete acidic or basic domains.


Figure 7: Secondary structure predictions for p230. Top panel, hydrophilicity profile of p230 using Kyte-Doolittle hydropathy scale with a window of 7(49) . Positive values denote hydrophilic regions that may be exposed on the outside of p230. Bottom panel, a summary of secondary structure predictions using the methods of Chou-Fasman (50, 51, 52, 53) (CF, light shaded bars) and Robson-Garnier(54, 55) (RG, dark shaded bars) with a hydrophilicity window size of 11 is shown, together with a composite where both Chou-Fasman and Robson-Garnier predictions are in agreement (CfRg, black shaded bars). The presence of a bar indicates regions of the protein predicted to form alpha-helices, beta-pleated sheet, or reverse turns by each of the methods used. These plots were generated using the MacVector program (International Biotechnologies Inc.).



The above features raised the possibility that p230 adopts a coiled-coil structure, stabilized by heptad repeats. A search for these structures was performed using the method of Lupas et al.(26) . The results of this analysis (Fig. 8a) reveal an extraordinarily high level of heptad repeats in domains 2 and 4, which predict a coiled-coiled structure with a high degree of confidence. Detailed sequence analysis of the longest of these regions is shown in Fig. 8b, which shows a run of 31 heptad repeats extending over 245 amino acids with four heptad frame-shifts. In common with various fibrous coiled-coil proteins(37, 38) , this region shows a high frequency of apolar residues in positions a (75%) and d (52%) of the heptads and the absence of acidic residues in these positions. The preference for leucine over isoleucine at the d position suggests that the protein has a dimeric rather than a trimeric quaternary structure(39, 40) . This region also shares with the fibrous proteins a high frequency (54.9%) of charged residues in the outer positions (b, c, e, f, and g), with only 14.3% of residues in these positions being apolar. However, in contrast to the fibrous proteins, this region has a relatively high number of lysine and arginine residues in the d position, which, together with the concomitant reduction in apolar residues at this position and the presence of other discontinuities including frameshifts and ``stutter residues,'' would be expected to confer marginal stability on the coiled-coil(36) .


Figure 8: A, prediction of coiled-coil segments of p230. The lower panel is a histogram of the probability of forming a coiled-coil structure according to the method of Lupas et al.(26) . p > 0.9 are significant. Bars above the histogram indicate which of the possible seven ``frames'' the heptad repeats follow for each region of the protein with P>0.5 for formation of a coiled-coil structure. The presence of numerous ``frameshifts'' provides evidence of discontinuities in the coiled coil structure (see text). B, primary sequence of the longest uninterrupted region of heptad repeats from p230. The amino acid sequence of p230 from position 1460 to 1704 is plotted to show the position of each residue within a heptad repeat of the form abcdefg(38) . Apolar residues at positions a and d are shown in boldface.



p230 has multiple consensus motifs for tyrosine phosphorylation, protein kinase C phosphorylation, casein kinase II phosphorylation, cAMP/cGMP phosphorylation, N-myristoylation, and N-glycosylation. It is not known at present how many of these motifs are utilized; however, p230 appears to be devoid of N-glycans(13) , consistent with location of the protein on the cytoplasmic face of Golgi membranes. p230 also contains between amino acids 538 and 546 the sequence ESLALEELEL, a motif found in otherwise diverse members of the granin (chromogranin/secretogranin) family of acidic proteins found in the secretory granules of neuroendocrine cells. A search of the translated GenBank(TM) data base revealed that in addition to chromogranins, this region and its immediate flanking sequence showed homology with a number of proteins involved in subcellular compartmentalization and motor functions, including flagellin, calnexin, the Golgi protein giantin, beta8-tubulin, neurofilament L, caldesmon, chromokinesin, and with the human microtubule-associated protein E-MAP-115. No known microtubule binding motifs were found in p230.

Chromosomal Localization

Using g5 clone as a probe, individual grains recorded on a cumulative idiogram revealed a significant accumulation of signal on a region of the short arm of human chromosome 6, assigning the probe to 6p12-22. Fig. 9a shows a diagrammatic presentation of the genomic distribution of grains observed in 70 metaphase spreads on one slide. An enlarged diagram of the grains recorded on chromosome 6, showing a peak at 6p12-22, together with photographs of a silver grain located at the specific site of hybridization, are shown in Fig. 9b.


Figure 9: Chromosomal localization of human p230 gene. A, diagram showing the grain distribution in 70 metaphase spreads on one slide following hybridization with ^3H-labeled DNA from clone g5; B, an enlargement of grains recorded on chromosome 6 together with photographs showing silver grains on G-banded chromosome 6.




DISCUSSION

The cDNA clones we have isolated encode the full-length p230 Golgi protein for the following reasons. First, autoantibodies, affinity-purified from clones g2, g5, g7, and g12, gave immunofluorescence staining of the Golgi apparatus of Hep2 cells. Second, rabbit antibodies raised against a bacterial fusion protein incorporating clone g5 not only stained the Golgi apparatus by immunofluorescence and immunoelectron microscopy, but also immunoblotted and immunoprecipitated a 230-kDa protein from HeLa cells (13) . Third, cDNAs of clones px1, z9, and z16 together span 7.7 kb, in agreement with the size of the mRNA obtained from Northern blots; and fourth, reverse transcripase PCR demonstrated that these three clones were derived from the same transcript.

The deduced amino acid sequence of p230 suggests a hydrophilic, modestly acidic protein capable of forming a dimeric coiled coil structure. While most (>90%) of the protein is predicted to form this structure, the extreme amino-terminal 130 amino acids and a segment between amino acids 239 and 270 are predicted to form compact structures consistent with globular regions. While it is well established that stable static coiled coils can serve as multimerization motifs in structural proteins, dynamic coiled-coil formation can play a central role in generation of conformational changes resulting in dramatic movements of one part of a protein relative to another. Such coiled-coil regions have been implicated in the function of the nonclaret disjunctional kinesin-related microtubule motor protein, which translocates on microtubules toward their minus ends and is required for proper chromosome segregation in Drosophila oocytes(41) . This protein has a central stalk region consisting of heptad repeats predicted to form coiled-coils. A mutant that lacks the amino-terminal third of the coiled-coil stalk exhibits partial loss of function, having a translocation velocity and torque generation similar to wild-type protein, but only partially rescues a null mutant for chromosome missegregation. A similar effect has been reported for a cytoplasmic myosin II protein partially deleted for its coiled-coil tail(42) . In this case, the mutant protein is expressed at a level comparable with the wild-type protein and translocates on actin filaments in vitro with the same velocity as wild-type protein, but in spite of this, exhibits frequent failure of cytokinesis in vivo. Of particular interest is the loss of function reported for partial deletions of the coiled-coil domain of the yeast Uso1 protein(43) , a protein involved in vesicular transport, where such mutations are temperature-sensitive lethal, resulting in a severe defect of endoplasmic reticulum to Golgi protein transport at the nonpermissive temperature. It has been suggested that a key structural feature of coiled-coils that participate in conformational changes is the presence of regions of marginal stability caused by discontinuities in heptad repeats of the coiled coil as a result of deletions, insertions, or out-of-frame residues(44) . The presence of extensive regions of this type in p230 raises the possibility of dynamic coiled-coil formation, which could play a role in regulation of multimerization or in induction of conformational changes in the protein. Dramatic conformational changes have been found to occur in other coiled-coil proteins involved in membrane fusion events (for review, see (44) ).

p230 also has a short region of homology to the conserved carboxyl-terminal domain of the granin (chromogranin/secretogranin) family of proteins, a diverse group of acidic proteins present in secretory granules of endocrine and neuroendocrine cells (for review, see (45) ). Granins are suggested to be precursors of several peptide hormones and to regulate proteolytic processing and selective aggregation of secretory proteins in the trans-Golgi network of neuroendocrine cells(46) . Only one short region at the carboxyl terminus is shared among the granins. This region bears the consensus sequence E(N/S)LX(A/D)X(D/E)XEL which is closely related to a sequence found in p230 (Fig. 6). A short region of the carboxyl terminus of chromogranin A, containing the granin motif, may be responsible for pH-regulated multimerization of the protein (47) and, together with the pH-dependent association of chromogranin A with integral membrane proteins of the secretory vesicle, suggests a role for chromogranins A in the sorting of these membrane proteins during vesicle biogenesis in the trans-Golgi network(48) . However, the relevance of this motif in p230 is unclear since p230 is orientated on the cytosolic side of Golgi membranes.

There is an increasing number of proteins implicated in vesicular transport that have extensive coiled-coil domains. These domains have potential for dynamic interactions associated with this highly complex process. The potential dynamic coiled-coil structure of p230, its localization to the trans-Golgi network, (^3)and sensitivity to brefeldin A (13) suggest that p230 may have a key role in vesicular transport from this distal Golgi compartment.


FOOTNOTES

*
This work was supported by grants from the Australian Research Council and a Nancy E. Pandergrast Research Fellowship of the Victorian Lupus Association. 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(TM)/EMBL Data Bank with accession number(s) U41740[GenBank].

§
A National Health and Medical Research Council Australian Postdoctoral Fellow.

To whom correspondence should be addressed: Dept. of Pathology and Immunology, Monash University Medical School, Prahran, Victoria 3181, Australia. Tel.: 613-9276-2713; Fax: 613-9529-6484; bht{at}cobra.path.monash.edu.au.

(^1)
The abbreviations used are: SNAPs, soluble N-ethylmaleimide-sensitive fusion protein attachment proteins; SNARE, SNAP receptors; PBS, phosphate-buffered saline; PCR, polymerase chain reaction.

(^2)
A. C. E. Knight, unpublished results.

(^3)
P. A. Gleeson and G. Griffiths, unpublished results.


ACKNOWLEDGEMENTS

We thank Rob Parton for critical reading of the manuscript and Fiona Matheson for technical assistance.


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