SEC7 Encodes an Unusual, High Molecular Weight Protein

THE JOURNAL OF BIOLOGICAL CHEMISTRY 0 1988 by The American Soeiety for Biochemistry and Molecular Biology, Inc.

Vol. 263, No. 24, Issue of August 25, , pp. 11711-11717,1988 Printed in U.S.A.

SEC7 Encodes an Unusual, High Molecular Weight Protein Required for Membrane Traffic from the Yeast Golgi Apparatus*

(Received for publication, April 1, 1988)

Tilman Achstetterg, Alex Franzusoffi, Charles Field, and Randy Schekman From the Department of Biochemistry, Uniuersity of California, Berkeley, California 94720

Saccharomyces cerevisiae with mutations at the sec7 locus are pleiotropically deficient in protein transport within the Golgi apparatus and proliferate a large array of Golgi cisternae at a restrictive growth temperature (37 OC). The SEC? gene and its product (Sec7p) have been evaluated by molecular cloning and sequence analysis. Two genes that allow sec7 mutant cells to grow at 37 O C are represented in wild-type yeast DNA libraries. A single copy of the authentic SEC7 gene permits growth of mutant cells, whereas the other gene suppresses growth deficiency only when expressed from a multicopy plasmid. The SEC7 gene is contained on a 8.4-kilobase pair SphI restriction fragment, portions of which hybridize to a single 6-kilobase pair mRNA. The gene is essential for yeast vegetative growth. DNA sequence analysis of this region detects a single open reading frame with the potential to encode a 2008-amino acid-long hydrophilic protein of 230 kDa. Putative Sec7p contains an unusual, highly charged acidic domain of 125 amino acids with 29% glutamate, 18% aspartate, and 21% serine. Within this region, stretches of 14 consecutive glutamate residues and 13 consecutive glutamatestaspartates are predicted. This domain in Sec7p may serve a structural role to interact with lipids or proteins on the cytoplasmic surface of the Golgi apparatus.

The pathways of intracellular protein traffic have been studied by a variety of approaches. Biochemical assays that detect the participation of soluble proteins have yielded im- portant clues concerning the mechanism of protein translocation across membranes (1, 2). Similar reactions that meas- ure intercompartmental transport of proteins show promise (3-5), but have not yet resulted in the identification of individual proteins that facilitate this process.

An alternative that may complement the biochemical ap- proach is the identification of gene products that are required at unique points in the secretory process. We have defined a

*This work was supported in part by Grants GM26755 and GM36881 from the National Institutes of Health. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertise- ment” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

to the GenBankTM/EMBL Data Bank with accession number(s) The nucleotide sequence(s) reported in this paper has been submitted

50391 8.

1988). This paper is dedicated to the memory of Charles Field (1947-

$ Supported by Max Kade Foundation Fellowship M480. Present address: Transgene S.A., 11 rue de Molsheim, F-67000 Strasbourg, France.

3 Supported by National Institutes of Health National Research Service Award 10673.

pathway of secretion in Saccharomyces cereuisioe by the iso- lation and characterization of a series of conditionally lethal mutations (6). At least 23 (SEC) genes are implicated in the process of intercompartmental protein transport (7, 8). Most of the sec mutations block transport of proteins from the endoplasmic reticulum to the Golgi apparatus or from mature secretory vesicles to the plasma membrane. Two mutations (see7 and secl4) block protein traffic within the Golgi apparatus (9, 10).

Mutations that define the sec7 locus exert a unique and dramatic effect on traffic of secretory, plasma membrane, vacuolar, and endocytic marker molecules (9-11). At a restrictive growth temperature (37 “C), sec7 mutant celIs accumulate multiple exaggerated Golgi-like membranes (5). Golgi cisternae collect in unusually large stacks when the incubation at 37 “C is conducted in media containing low concentrations of glucose. Secretory glycoproteins accumulate within these cisternae (8, 9) and are exported when cells are returned to a permissive temperature (24 “C).

Examination of the secreted pheromone a-factor accumu- lated in sec7 cells at the nonpermissive temperature shows that maturation of oligosaccharide chains has occurred, but that the block precedes endoproteolytic digestion of the precursor polypeptide (12, 13). However, the vacuolar protein carboxypeptidase Y exhibits both a defect in the maturation of its oligosaccharide chains and endoproteolytic cleavage of the precursor polypeptide as a result of the sec7 block (10). Hence, the SEC7 gene product (Sec’lp) may regulate protein traffic from various compartments of the Golgi apparatus.

In this report, we describe the molecular cloning and analysis of the SEC7 gene.

EXPERIMENTAL PROCEDURES

Strains, Plasmids, Growth Conditions, and Materials-The yeast and bacterial strains used in this study are listed in Table I. Esche- richia coli plasmid pUC18 and the M13-derived versions of M13mp18 and M13mp19 were used as described (14). Yeast centromeric plasmids YCp50 (15), pSEYc58 (16), and the high copy number (2p- based) plasmids YEpl3 (17) and pCF35 were used as E. coli-yeast shuttle vectors. Integrating plasmid YIp5 was used as described (18). A yeast genomic DNA library, constructed with a partial S a d A restriction enzyme digest inserted into the BamHI site of YCp50, was provided by P. Novick (Yale University, New Haven, CT) and M. Rose (Princeton University, Princeton, NJ) (19). The cDNA expression library of McKnight and McConaughy (26) was used as before.

Yeast cells were grown in YPD medium (1% yeast extract, 2% peptone, 5% glucose). Their growth was monitored by optical density at 600 nm as described (20).

Restriction endonucleases, DNA-modifying enzymes, and DNA polymerase were from Bethesda Research Laboratories; ATP and deoxynucleoside triphosphates were from Sigma; dideoxynucleoside triphosphates were from Pharmacia LKB Biotechnology Inc.; and [LY-~*P]~CTP and U - ~ ~ S - ~ C T P were from Amersham Corp. Lyticase was prepared as described (21).

Moleculur Cloning of SEC7”Restriction endonuclease digestions and T4 DNA ligase reactions were carried out according to the

11711

11712 SEC7 and Golgi Traffic

TABLE I Yeast and bacterial strains

Strains Genotype Source

Yeast X2180-1B MA Ta gal2 YGSC" SF821-8A MATa sec7-1 his4-580" urd-52 ku2-3 This study

SF911- leu2-112 trpl-289 Mata sec7-4 his4-580" urd-52 leu2-3 This study

leu2-112 trpl-289 13D

SEY2101 MATa urd-52 leu2-3 leu2-112 ade2-1 S. Emr

S2615B MATa hom2 arolC leul adel ade5 gal2 YGSC" S U C ~ - A ~

TAY67-2D MATa urd-52 leul leu2-3 leu2-112 This study

TAYD68 hom2 arolC d e 2 d e 5 suc2-A9 MATalMATa sec7"1/SEC7 urd-52/urd-52 This study

HOM2/hom2 AROIC/arol C his4-58On/HIS4 ADE2/adeBADE5/ade5 trpl-289/TRPI

le~2-3/leu2-3 le~2-112/le~2-112

S U C ~ / S U C ~ - A ~ Bacterial

MC1061 F-araD139 A(araABOlC-leu)7679 Alacx74 M. J. Casadaban

"21061 MC1061 containing a chromosomal Tn5 R. Foster and J. Rine [chr::Tn5] insertion MV1193 A(1ac-proA,B) thi supE A(srl-recA)306: J. Viera

TG1 A(1ac-proA,B) thi supE hsdD5 (F':trad 36 T. Gibson

SElO

JM103 A(1ac pro) thi strA supE endA scbB hsdR- J. Messing

galU- galK- rpsL- hsdR-

TnlO (tet') [F':trad36proA,B lacIqZAM15]

proA,B lacIqZAM15]

ZAM15)

F'traD36 proAB lacIqZAM15

pyrF:Tn5 ara(A1ac pro) rpsL- thi(480dlac- S. Emr

' Yeast Genetic Stock Center (University of California, Berkely, CA).

manufacturer's instructions. Reactions with T4 DNA polymerase and E. coli DNA polymerase I (Klenow fragment) were performed as described by Maniatis et al. (22). Techniques of plasmid preparation, agarose gel electrophoresis, and DNA transformations of E. coli and S. cereuisiue were as described (22-24).

sec7 yeast (strain SF821-8A) was grown to ODWnm = 1-2, converted to spheroplasts, and transformed with a genomic DNA library (25) or a cDNA expression library (26). Transformants were selected on minimal medium lacking leucine (for the genomic library) or tryptophan (for the cDNA library). After 2 days, at which time transformants began to appear, plates were transferred to the sec restrictive temperature (38 "C). Colonies that continued to grow were selected and replated, and plasmid DNA was isolated from each. Yeast plasmid DNA was used to transform E. coli MC1061. Plasmid DNA was isolated from individual transformants, and complementation of both the growth defect and the auxotrophic requirement was confirmed by transformation of the original yeast host.

Plasmid pCF36, obtained from the genomic centromere library (19), contained a 12-kb' insert carrying the authentic SEC7gene (Fig. lA). pTA33 contained a 8.4-kb SphI fragment (Fig. lA, fragment A) that included the entire SEC7 gene in pUC18. Subsequent subcloning was done from pTA33. YEpTA67 and pTAYc66 contained fragment A in YEpl3 and YCp50, respectively. YEpTA65 and pTAYc64 contained a 7.5-kb PstI fragment (Fig. l A , fragment E ) , missing only the 3' end of SEC7, in pCF35 and pSEYc58, respectively. YEpTA34 and pTAYc35 carried a 7.2-kb XhoI-BamHI fragment (Fig. lA, fragment C), missing potential regulatory sequences upstream from the coding region, in pCF35 and YCp50, respectively. pTAYcl7 contained a 5.5- kb EcoRI fragment, including most of the coding region of SEC7, in YCp50 (Fig. lA, fragment D).

Plasmid p334, from the cDNA library, contained a 1.4-kb insert with a gene that, when overexpressed, suppressed the growth deficiency of sec7 mutant cells (Fig. 1B).

Transposon-mediated mutagenesis of SEC7 was performed as described by Bernstein et al. (20). Five independent Tn5 insertions into pCF36 were obtained.

For disruption of the SEC7 chromosomal locus, a 4.6-kb EcoRI internal fragment was subcloned into pUC18. A deletion within this

' The abbreviation used is: kb, kilobase pair.

insert was created by cleavage with ChI. The resultant cohesive termini were filled in by treatment with DNA polymerase (Klenow fragment) and deoxynucleoside triphosphates. A 2.2-kb BglII fragment containing the LEU2 gene (27) was isolated from YEpl3, and the termini were filled in as described above. This insert was ligated to the pUC18 plasmid described above to create YIpTA58. The direction of transcription of the LEU2 gene with respect to the disrupted SEC7 gene is indicated in Fig. 2. YIpTA58 was cleaved with PuuII to produce a linear fragment with 230 base pairs of pUC18 at one end and 500 base pairs of SEC7 at the other terminus. This fragment was transformed into a diploid yeast strain (TAYD68) heterozygous at the SEC7 locus. Leu+ transformants were obtained at a frequency of about 9/Fg of DNA/lOs spheroplasts.

Southern hybridization (22) was performed on DNA isolated from untransformed and transformed strains (27).

Northern hybridization (28) analysis of SEC7 mRNA was performed with a 0.7-kb EcoRI fragment (Fig. 2, fragment A) and a 0.6- kb PstI fragment (Fig. 2, fragment C) that were subcloned into pUC18 and with a 3.8-kb BglIII-EcoRI fragment (Fig. 2, fragment B ) that was subcloned into YIp5. Radioactive probes were produced by nick translation. Poly(A)+ mRNA was prepared from wild-type X2180-1B yeast as described (22).

A strand-specific probe was obtained by inserting a 2-kb BglII- EcoRV internal fragment of SEC7 into the replicative form of E. coli phage derivatives Ml3mplS and M13mp19 that had been cleaved with BamHI and S m I nucleases (13). Phage produced by cells infected with these recombinants were used as a source of template for probe synthesis primed by a 13-mer oligodeoxynucleotide (New England BioLabs) as described by the manufacturer.

DNA Sequence Analysis-The SphI DNA fragment (8.5 kb) from pCF36 (Fig. 1) was purified and converted to concatemeric DNA by incubation with DNA ligase as described (29). Random fragments (700-1000 base pairs) were generated by sonication. Samples chilled on ice were sonicated four times for 15 s using a Branson sonifier at 50-watt power. Fragments were converted to complete duplexes with the Klenow fragment of DNA polymerase as described (22). The resulting blunt end fragments were ligated into Ml3mplO that had been restricted by S m I and dephosphorylated by alkaline phospha- tase (Pharmacia LKB Biotechnology Inc.). Recombinants were prop- agated in E. coli strain TG-1. Template preparation and DNA se-

SEC7 and Golgi Traffic 11713

A E N IY

I +I- C L I + D J I

B

FIG. 1. Restriction map of plasmids pCF36 (A) and p334 (B) and fragments of pCF36 subcloned into different vectors. A, pCF36 contains a 12-kb insert in YCp50; B, p334 contains a 1.2- kb insert. The black arrows describe the approximate ends of the coding region of SEC7 (for details, see "Results and Discussion"). Pu, PuuII; X, X b I ; Ps, PstI; R l , EcoRI; Sp, SphI; B, BglII; C, ClaI; RV, EcoRV; Ba, BamHI; H, HindIII. V, sites where Tn5 transpositions have occurred. The complementation table refers to the ability of transformed multicopy plasmids carrying the designated inserts to restore growth to sec7-1 cells at the nonpermissive temperature.

quencing by the method of Sanger et al. (30) using a-35S-dCTP were performed as described (31). M13 primer (17-mer) for sequencing was provided by Bruce Malcolm (this Department). DNA sequences were compiled and analyzed with Intelligenetics software programs. The SEC7 open reading frame sequence was compared to the Dayhoff Protein Data Bank (NBRF) by the program of Lipman and Pearson (32) as implemented on BIONET.

RESULTS AND DISCUSSION

Cloning of the SEC7 Gene-The SEC7 gene was cloned by complementation of the temperature-sensitive sec7-1 defect. Mutant cells (strain SF821-8A) were transformed separately with a yeast genomic and a yeast cDNA expression library. Transformants were selected for growth on medium lacking either uracil or tryptophan and for growth at 38 "C, the restrictive temperature for the sec7-1 mutation. From the cDNA library, -40,000 Trp' colonies were obtained, of which

two were also thermoresistant Ts'. These two cDNA clones were identical by restriction enzyme analysis (p334 cDNA clone; Fig. 1B). The cDNA clone complemented only the sec7- 1 mutation, but not another allele, sec7-4. Furthermore, the SphI-HindIII fragment of p334, which carried the complementing gene, failed to restore Ts' growth to sec7-1 cells when inserted into the single copy YCp50 centromere plasmid. From this, we suspected that p334 expressed an allele-specific suppressor function, but only when overproduced. Independ- ent genetic evidence showed that this gene integrated at a chromosomal locus separate from the sec7 locus. For these reasons, further investigation of the nature of the p334 gene was postponed.

The genomic library yielded -50,000 Ura+ colonies, of which five were also Ts+. These five clones were identical by restriction enzyme analysis (pCF36 genomic clone; Fig. lA), but completely distinct from the cDNA clones described above. The genomic clone complemented both the sec7-1 and sec7-4 mutations. To identify the region of the 12-kb insert that carried complementing activity, plasmid pCF36 was mu- tagenized by random insertion of the transposon Tn5. Five independent isolates of pCF36 with a Tn5 insertion in the yeast genomic sequence were obtained; four of these insertion events inactivated pCF36 complementation of sec7-1 (Fig. 1, Tn5-13, -14, -22, and -26). Restriction enzyme analysis of the position of transposon insertion indicated that the SEC7 complementing activity extended close to the rightmost end point of the yeast insert as depicted in Fig, 1. Additional Tn5 insertions confirmed the size of the gene and marked the 5' boundary of the SEC7 gene (data not shown).

A subclone of pCF36 that exhibited SEC7 complementing activity was a 8.5-kb SphI-SphI fragment (Fig. lA, fragment A). This fragment introduced either on a centromere plasmid (pTAYc66) or on a multicopy plasmid (YEpTA65) restored normal growth to sec7 mutant cells at 38 "C. Similar results were seen with fragment C in single copy or multicopy plasmids (pTAYc35 and YEpTA65, respectively). Partial complementation was obtained with fragment B, whereas fragment D was unable to restore Ts+ growh to sec7 cells.

Analysis of SEC7 mRNA by Northern hybridization confirmed the large size of the gene. Fragment B (Fig. 2) hybrid- ized to a unique 6-kb species represented in poly(A)+ mRNA from a wild-type strain (Fig. 3). Fragments A and C (Fig. 2) detected the same species. By comparison with an internal standard, the SEC7 mRNA appeared to be about 20% as abundant as URA3 mRNA, or perhaps about five copies/cell.

SEC7 Encodes an Essential Gene-Homologous integration was used to demonstrate that pCF36 contained the authentic SEC7 gene. The yeast LEU2 gene was introduced in place of a 2.2-kb ChI-ChI fragment and then excised as a 5.5-kb EcoRI-EcoRI fragment (Fig. 2, fragment E) so that SEC7 sequences flanked the LEU2 gene. This linear molecule was introduced into the sec7/SEC7 heterozygous diploid (strain TAYD68) by spheroplast transformation. The SEC7 locus is closely linked to the arolC and horn2 loci on the right arm of

FIG. 2. Analysis of the SEC7 SP gene. The black arrow represents the SEC7 gene and the direction of transcription. DNA restriction fragments A- A 1 B I 1 c D were used to make nick-translated probes for Northern hybridization ' lkb ' I analysis of SEC7 mRNA. Fragment E was used as a linearized fragment for gene replacement and disruption of the

tails, see "Experimental Procedures").

D I

SEC7 locus on the chromosome (for de- Y*"R E ?

LEU2


kb

9.4 - 6-6- - SEC7 4.4 -

2.3 - 2.0 -

e URA3

0.6 -

FIG. 3. SEC7 codes for a 6-kb transcript. Northern hybridization analysis of SEC7 mRNA prepared from wild-type X2180-1B yeast cells was performed as described under "Experimental Proce- dures." The 0.7-kb EcoRI fragment (Fig. 2, fragment A ) from SECI DNA was used to probe the blot. A URA3 DNA probe was used as an internal standard for size and quantitation.

chromosome IV (33): Integration of the linear construct into the chromosome is directed by homologous recombination with the ends of the molecule. Out of 45 Leu' transformants, 17 exhibited temperature-sensitive growth, suggesting gene replacement of the wild-type SEC7 locus. Restriction enzyme analysis and Southern hybridization with probes derived from the undeleted segment of cloned SEC7 demonstrated integration of LEU2 at identical loci in the thermoresistant (Ts') and thermosensitive (ts-) diploid transformants (data not shown). Further support that integration occurred at the SEC7 (or sec7) locus was obtained from the characterization of spores derived from stable Ts' and ts- diploid transformants. The Ts' diploid generated tetrads (17 analyzed) which segregated two viable Ts' and two dead spores. Likewise, the ts- diploid generated tetrads (22 analyzed) consisting of two viable ts- and two dead spores. All viable spores were Leu-, demonstrating that integration had disrupted the SEC (or sec) locus and generated a lethal phenotype in the null mutant spores. Segregation of the other markers (arolc and b m 2 ) confirmed tight linkage to the SEC locus, whereas other loci (MAT, TRPl, ADE2, HIS4) segregated independently of the lethal phenotype. We conclude that integration of fragment E (Fig. 2) had occurred at the SEC7 locus and that disruption of this gene prevented spore germination. Hence, SEC7 is an essential gene for growth in yeast.

DNA Sequence Analysis of SEC7-Random cloning was used to sequence most the the SphI-SphI fragment (8.5 kb) of pCF36. Two short stretches of DNA that were not found among the (377) sequenced random clones were subcloned into M13 by direct procedures. The information in Fig. 4 was determined by sequencing both strands entirely.

Computer analysis of the SEC7 DNA sequence revealed a single open reading frame encoding a protein of 2008 amino acid residues (Fig. 4), with a predicted molecular weight of 227,885. The position of this open reading frame within the 8.5-kb fragment is shown by the black arrows in Fig. 1. Translation is predicted to start at an ATG 11 bp upstream from the second of two closely spaced EcoRI sites.

Potential transcriptional start sites, the TATA sequences

C. Field, unpublished data.

(34), were noted at nucleotides -301, -152, and -8 relative to the putative translation start codon. A number of yeast genes that encode abundant mRNAs have transcription start sites at or very near the sequence CAAG, and a CT-rich block is often found upstream of this sequence (35). This pattern of nucleotides was noted upstream of the distal and proximal (but not the medial) TATA boxes of the 5'-SEC7 noncoding region. The relevance of these sequences to SEC7 transcription is not clear inasmuch as Northern hybridization showed SEC7 mRNA to be less abundant than URA3 mRNA (Fig. 3). This low level may relate to stability of an unusually large mRNA or to a low rate of transcription. By comparison with well-characterized yeast genes, the TATA sequence at position -152 approximates the normal position relative to the start codon.

The consensus sequence ANNAUGNNU has been proposed as a site that marks efficient translation initiation in yeast (36). The ATG at position +1 (CUGAUGUCU) in Sec7p does not fulfill the predicted consensus requirements.

SEC7 appears not to contain introns. Consensus sequences for splice junctions were not detected. Furthermore, the con- tinuity of the open reading frame and correspondence in size to the mRNA size suggest the absence of noncoding interven- ing sequences.

A termination codon (TAA) was observed 333 nucleotides downstream from the distal PstI restriction site, and an additional 286 nucleotides were sequenced. This latter region does not contain typical yeast transcription termination sig- nals (37,38). Whereas the first 100 nucleotides of this putative 3'-noncoding region have the usual AT-rich character (69%), the remaining 186 nucleotides are GC-rich (64%). The latter may correspond to vector sequences since the distal SphI restriction site is contained in the plasmid (pCF35) used to express the genomic library.

Sec7p Sequence Analysis-The large open reading frame of the SECI sequence was used to scan the NBRF Protein Data Bank for homologies. Analysis was performed by the FASTP program (32) using the entire or smaller portions of the predicted sequence. No significant homologies to any regis- tered protein sequences were noted. Domains characteristic of nucleotide-binding, calcium-binding, or protein kinase activity also were not detected.

Eight potential N-linked glycosylation sites (Asn-X-Thr) were noted in the Sec7p sequence. These sites, if used, would be diagnostic of a glycoprotein within the secretory pathway. Since Sec7p is required for membrane traffic from the Golgi apparatus, the protein may function from within the organ- elles of the secretory pathway. The N-terminal region of Sec7p was not predicted to be hydrophobic, as would be expected if Sec7p contained a signal sequence for translocation across the endoplasmic reticulum. Hydropathy analysis (39) of Sec7p predicted a strongly hydrophilic character for the polypeptide. Only one region of the sequence approximates the hydrophobic structure of a membrane anchor domain. This region, starting at amino acid 1917, extends for 19 residues and comprises 2 charged, 7 polar, and 10 hydrophobic amino acids. It seems unlikely, therefore, that SEC7 encodes either a membrane-spanning protein or a soluble luminal protein. We suspect that Sec7p resides in cytosol or is associated with the cytoplasmic surface of a membrane and therefore does not utilize any of the eight potential N-linked glycosylation sites.

One striking feature of the predicted Sec7p was the highly acidic domain near the amino terminus (Fig. 5). Beginning at residue 89, 25 of the next 29 amino acids (84%) are acidic. The segment from positions 89 to 213 has a predicted PI of 3.0 and consists of 47% acidic residues, including stretches of


if5 T h r P r o G l u G l u T h i G l u ADP Thr A m A s p L y s A r q H i 9 A s p A s p G l u G i y G l u A s p G l y ASP G l u A s p G l u A s p G l u A s p G l u A s p G l u A s p G l u A s p A s " G l y A s p AAA ACG CCC GAA GAA ACG GAA GAT ACA AAT GAC AAA CGC CAT GAC GAT GAA GGT GAA GAT GGA GAT GAA GAT GAA GAT GAA GAT GAA GAT GAA GAT GAA GAC AAT GGG GAC

80 90 100 1 1 0

G l u A s p A s p G l u A s p V a l A s p Ser Ser S e r Ser G l u T h r Ser Ser G l u A s p G l y G l u ASP S e r G l u Ser V a l Ser G l y G l u Ser T h r G l u Ser Ser Ser G l y G l u ASP G l u 1 2 0 130 1 4 0

GAG GAT GAT GAG GAC GTG GAT AGC AGT ACT TCC GAA ACC AGT TCC GAA GAC GGG GAA GAT TCT GAA TCR GTC TCA GGG GAA AGT ACT GAG AGT AGC TCT GGA GAA GAT GAA

150 G l u Sei: A s p G l u S e c A s p G l y A m T h i Sex A s n Ser Ser S e r G l y A s p G l u ser G i y S e r G l u G I " G l u G l u G l u G l u G l u G l u G l u G l u G I " G l u G l u G l u AS" A l a G l y GAA TCT GAT GAA AGC GAT GGA AAT ACA TCA AAC AGC TCC TCT GGT GAT GAA AGC GGG TCA GAA GAA GAA GAA GAA GAA GAA GAA GAA GAA GAA GAA GAG G M AAT GCT GGC

160 1 7 0 1 8 0

1 9 0 G l u P r o Ser Ile A l a !lis G l n A s p Ser V a l P r o T h r A s " A 3 p Ser T h r A l a P r o A r 9 S e r T h r H i 3 T h r A r q AS" l l e Ser L e u Ser Ser A s n G l y Ser A s n T h r AS" S e r GAA CCC AGT ATC GCT CAT CAA GAT AGT GTT CCC ACC AAC GAT TCT ACT GCC CCA AGG TCT ACC CAT ACG AGG AAC ATA TCA CTA TCT TCA AAT GGT TCG M C ACA AAC TCA

2 0 0 210 220

T h r Ile Ile L e u V a l L y a T h r T h r L e u G I " T h r I l e L e u A s n A s p L y l ADP Ile L y a L y s A 3 n Ser A s n A l a G l n L y s A l a I l e G i u A r q T h r L e u G i n L y a P h e L y s Glu ACC ATA ATT TTA GTG AAA ACT ACG TTG GAG ACA ATT TTA AAT GAT AAG GAC ATT AAA AAG AAT TCG AAT GCT CAG AAG GCT ATC GAA AGG ACA TTA CAA AAA TTT AAG GAA

2 3 0 2 4 6 2 5 0

2 6 0 P h e A s p P r o G l n T h r T h r R a n A s n P r o Ha$ T y r V a l ASP Ser I l e Leu V a l Phe G l u A l a L e u A r q A l a Ser Cy9 A c q T h r L y s Ser Ser L y S V a l G l n Ser L e u A l a L e u TTT GAT CCG CAA ACC ACG AAT AAC CCA CAT TAC GTG GAT TCA ATA CTA GTA TTC GAA GCA CTA AGG GCA AGC TGT CGT ACC AAA TCC TCC AAA GTT CAA AGT TTG GCT TTA

2 7 0 2 8 0 29"

300 AIP C y 5 L e u Ser L y s Leu Phe Ser Phe Arq Ser L e u ADP G l u T h r Leu L e u V a l A s " P r o P r o A s p Ser L e u A l a Ser A s " A 3 p G l n A r g G l n ASP A l a A l a A s p G l y lle GAT TGC CTG TCA AAA TTG TTT TCT TTT AGA TCG CTA GAC GAG ACC CTG TTA GTG AAT CCA CCC GAT I C T TTA GCC TCT AAT GAT CAA CGA CAA GAT GCT GCC GAT GGA ATA

3 1 0 3 2 0 3 3 0

3 4 c T h r P r o P r o P r o L y s G l n L y 5 I l e I l e A s p A l a A l a I l e ASP T l r Ile Ser ASP Cy9 Phe G l n G l y G l u G l y T h r A a p ASP A r g V a l G l u L e u G i n Ile V a l A r g A l a L e u

3 5 0 3 6 0 3 1 0

ACG CCG CCT CCA AAA CAA AAA ATT ATA GAC GCT GCA ATT GAT ACT ATT TCA GAC TGT TTT CAA GGT GAA GGC ACT GAT GAC CGT GTG GAA CTA CAA ATC GTT AGA GCT TTA

Ser Ser C y s I l e L e u G I 0 G l u A s p S e r Ser Sei L e u C y 4 H13 G l y A l a Ser L e u L e u L y s A l a Ile A ~ r g T h r I l e T y r As11 V a l L e o L e u Phe Cy3 L e u A m P r o S e r A s n TCT AGT TGC ATT TTA GAA GAG GAT TCA AGT TCT TTA TGC CAC GGT GCT TCC TTG CTA AAG GCT ATC AGA ACA ATC TAC AAC GTT TTG CTC TTT TGC TTG AAC CCA TCC AAT

380 390 400

410 G l n G l y I l e A l a G l n A l a T h r L e u T h r G l n I l e Ile Ser Ser V a l T y r A3p L y l Ile A s p L e u L y s G l n Ser T h r Ser Ser A l a V a l Ser L e u Ser T h r LyS As" H l 3 G l n CAA GGT ATT GCA CAG GCG ACC TTG ACA CAA ATT ATT AGT TCG GTG TAT GAT AAA ATC GAT CTC AAA CAA AGT ACC TCT TCA GCA GTA TCC TTA TCG ACA AAA AAT CAT CAA

4 2 0 430 4 4 0

450 G l n G l n Ser A l a l i e G l u L e u S e r G l u A l a Ser G l u A i r n A l a G l u T h r P r o A l a P r o L e u T h r L e u G l u A m Met A s p L y 3 L e u A 3 n A 3 p A s p G l u G l u A r q L e u M e t A3p CAA CAA TCC GCC ATA GAA CTT TCC GAG GCT TCT GAA AAT GCA GAA ACG CCG GCT CCT TTA ACT CTG GAA AAT ATG GAT AAG TTG AAT GAC GAT GAG GAA AGA CTA ATG GAC

4 6 0 4 1 0 a a o

A l a G l n G l n P r o A s p Ser Ile A l a I l e T h r A s " G l n A s p L e u A l a V a l L y 5 A s p A l a P h e L e u V a l P h e A r q V a l Met A l a L y s I l e C y s A l a L y 9 P r o L e u G l u T h r G l u 4 9 0 5 0 0 510

GCT CAA CAG CCT GAT TCT ATC GCC ATA ACT AAC C M GAT CTA GCT GTT AAA GAT GCG TTC TTA GTG TTT AGA GTC ATG GCG AAA ATA TGT GCT AAA CCT TTG GAA ACA GAA

L e u A s p M e t A r q Ser H i s A l a V a l A r 9 Ser L y s Leu L e u Ser Leu H l b I l e I l e T y r Ser I l e Ile L y s ASP H i 3 I l e A s p V a l P h e L e u Ser H 1 S A a n lie Phe Leu P r o CTC GAC ATG AGG TCA CAT GCC GTC AGG TCA AAG CTT TTA TCT CTT CAC ATC ATT TAC TCT ATT ATC AAA GAT CAT ATT GAC GTA TTC CTT TCC CAC AAC ATT TTT CTA CCA

5 2 c 5 3 0 5 4 0 5 5 0

5 6 0 G l y L y 9 G l u A r q V a l Cys Phe l l e A s p Ser Ile A r q G I " T y r L e u A r q Leu V a l L e u Ser A r 9 A s n A l a A l a Ser Pro L e u A l a P r o V a l P h e G1U V a l T h r L e u G l u Ile GGA AAG GAG CGT GTG TGC TTT ATC GAT TCA ATT A i A CAA TAT TTA CGT CTT GTT TTA TCA AGG AAT GCT GCC TCG CCT CTA GCT CCA GTT TTC GAG GTT ACT TTA GAA A T T

5 1 0 5 8 0 5 9 0

Mer T r p L e u Leu Ile A l a A 5 0 L e u A c q A l a A s p P h e V a l L y 3 G l u I l e P r o V a l P h e Leu T h i G l u I l e T y r P h e P r o I l e S e r Glu L e u T h r T h r S e r T h r Ser G l n G l n ATG TGG CTA TTG ATT GCT AAT TTG AGA GCA GAT TTC GTG AAG GAA ATT CCA GTT TTT TTA ACA GAA ATC TAC TTC CCC ATT TCA GAA TTA ACC ACT TCC ACC TCC CAA CAA

6 0 0 6 1 0 6 2 0

6 3 0 L y 5 A r q T y r P h e L e u S e r V a l Ile G l n A i r 9 Ile C y 3 A s n A s p P r o A r 9 T h r L e u V a l G l u P h e T y r L e u A s n T y r A s p Cy, Ais" P r o G l y M e t P r o A 5 0 V a l Met G l u I l e AAG AGA TAT TTT TTA AGT GTT ATT CAA CGA ATT TGT AAC GAC CCA AGA ACT TTA GTT GAA TTT TAC TTG AAT TAT GAT TGT AAC CCT GGA ATG CCI\ AAT GTA ATG GAA ATA

6 4 0 6 5 0 6 6 0

6 1 0 T h r V a l ASP T y r L e u T h r A r 9 L e u A l a L e u T h r A r q V a l G l u I l e T h r G l n T h r G l n A r q Ser T y r T y r A a p G l u G ln Ile Ser L y 3 Ser L e u S e l T h r T y r A 3 n Phe Ser ACT GTT GAT TAT TTG ACA AGA TTG GCT TTA ACT AGG GTG GAA ATT ACT CAG ACT CAA AGA TCA TAC TAC GAT GAA CAA ATA TCA AAA TCC CTG TCC ACC TAC M T TTC TCA

6 8 0 6 9 0 700

G l n L e u P r o L e u L e u T h r Ser Sei A s " L e u Ser Ser Ser P r o A s p V a l G l y G l n V a l A s n L e u L e u P h e P r o L e u A s p P h e A l a L e u L y S Met V a l Ser L e u AS" Cy3 I i e 710 7 2 0 1 3 0 7 4 0

CAA TTG CCC CTT TTG ACT TCT TCC AAC TTG TCT TCT AGT CCT GAT GTT GGT CAA GTC AAT TTA CTT TTT CCA CTT GAT TTT GCT CTC AAA ATG GTG TCA CTA AAT TGC ATT

V a l S e r V a l L e u A r q Ser L e u Ser Ser T r p A l a H i 9 L y s A l a L e u A s " P r o A s n T h r H i s T h c A l a A s n L y s V a l L e u Leu R a n T h r T h r Ser Sex A l a A r q G l n G l u Ser GTG TCA GTT TTG CGT TCC TTA AGC TCA TGG GCT CAC AAA GCT TTA AAT CCA AAC ACA CAC ACT GCT AAT AAG GTA TTA CTA AAC ACA ACA TCT TCA GCT CGT CAA GAA TCT

7 5 0 760 7 1 0

1 8 0 A r g S e r Ser L e u S e r A s n A s p V a l A i q Ser Ser I l e Mec T h r Ser A s " A s p A s p P h e L y s P r o T h r T y r G l u ASP G l u G l u Ser A r g Ser L e u Ser Ser G i n A s n I l e A s p AGA TCA TCT TTG AGT AAC GAC GTA AGG TCT TCT ATT ATG ACA AGT AAT GAT GAC TTC AAA CCA ACA TAT GAA GAC GAA GAA AGC AGA TCA TTG AGC AGT CAA AAC AT" GAC

7 9 0 8 0 0 8 1 0

a z o A l a A s p A s p P r o T h r G l n P h e GI" A s n Leu LyS L e u A r q L y S T h r A l a L e u Ser G l u C y s Ile A l a Ile P h e A 3 0 A s n L y S P r o Ly.3 L y S A l a I l e P I 0 V a l Leu lie Lyi r GCA GAC GAC CCC ACA CAA TTT GAA AAT TTG AAA CTA AGG AAA ACT GCT TTA TCG GAA TGT ATT GCT ATT TTC AAC AAT AAA CCC AAG AAA GCT ATT CCA GTA CTT ATT AAA

8 3 0 8 4 0 a 5 0

8 6 0 L y 5 G l y Phe Leu Ly3 A s p A l p Ser P r o I l e Ser I l e A l a L y S T l p L e v L e u G l u T h r G l u G l y L e u A s p M e t A l a A l a V a l G l y A 3 p T y r L e u G l y G l u G l y ASP A s p L y s AAA GGA TTT TTG AAA GAT GAT TCT CCA .4TT TCC ATT GCC AAG TGG TTA TTA GAA ACA GAA GGC CTC GAC ATG GCC GCT GTT GGG GAT TAT CTA GGC GAA GGA GAC GAC AAG

a 7 0 a a o

8 9 0 As" Ile A l a I l e M e t H i 9 A l a Phe Val ASP G l u P h e ASP Phe T h r G l y Met S e r I l e V a l A 3 p A l a L e u A r q Ser Phe L e u G l n Sei Phe A r q L e u P I 0 G l y G1U G l y G l n AAC ATC GCT ATA ATG CAC OCA TTT GTT GAT GAG TTT GAC TTC ACT GGT ATG TCC ATT GTT GAC GCA TTA AGG TCA TTT TTA CAA ACT TTC AGA TTG CCT GGA G M GGT CAA

900 9 1 0 9 2 0

9 3 0 L y s I l e A 9 p A r q P h e Met L e u L y s P h e A l a G l u A r q ? h e V a l A s p Gl? As" P r o G l y V a l P h e Ser L y s A l a A s p T h r A l a Tyr V a l L e u Ser T y r Ser L e u Ile Met Leu AAA ATT GAC AGA TTC ATG CTG AAA TTT GCG GAA AGA TTT GTG GAC CAA AAC CCC GGA GTC TTT TCA AAG GCG GAT ACT GCA TAT GTG CTT TCG T I T TCT TTG ATC ATG TTG

9 4 0 9 5 0 960

~ s n T h r A z p L e u HIS Ser Ser G l n Ile L y 3 A m L y s Mer Sei L e u G l n G l u P h e Leu G l u A s n A s " G l u G l y Ile h3p A m G l y A r g ASP L e u P r o A q A l p P h e L e u G l u AAT ACT GAT TTA CAT TCG TCA CAA ATC AAA AAT AAA ATG TCT TTA CAA GAG TTT TTA GAA AAC AAC GAA GGT ATT GAC AAC GGA AGA GAT TTA CCA AGA GAC TTC TTG GAA

910 9 8 0 9 9 0

FIG. 4. Sequence of the SEC7 gene. The complete sequence of the SEC7 gene as well as 724 nucleotides of flanking DNA are presented. The sequence is shown only for the coding strand of the gene. The TATA consensus sequences in the 5'-flanking DNA are underlined. The predicted Sec7p open reading frame, starting at nucleotide 440, is shown above the corresponding nucleotide sequence. Residue number is displayed in the open reading frame. Nucleotide number is shown for flanking DNA sequence.


1000 G i y Leu P h e A 4 n G i u I l e A l a A s n A s n G l u lie L y s L e u Ile Ser G l u G l n H i 3 G i n A l a Met L e u S e r G l y A s p T h r A a n L e u Val G l o G l n P r o A l a I l e C y 3 P h e G l n GGT TTG TTC M C GAA ATT GCT AAC AAT GAA ATC AAG TTA ATT TCT GAA CAG CAT CAG GCA ATG CTT TCA GGT GAT ACC AAT CTT GTC CAA CAA CCA GCA ATC TGC TTT CAA

1010 1020 1030

1040 Leu P h e A s n Ser A r q A S P L e u T h r A r q G l u A l a T y r A s n G l n Val Ser L y g G 1 u I l e Ser Ser L y s T h r G l u L e u Val P h e L y s A s n L e u A s " L y g A s n L y 3 G l y G l y p r o CTC TTT AAT TCT CGT GAT TTG ACA AGA GAG GCA TAT M T CAA GTC TCA AAA GAA ATT TCA TCT AAA ACG GAA TTA GTC TTT M G AAT TTA AAC AAA AAT AAA GGA GGc CCA

1 0 5 0 1060 1070

A s p Val T y r T y r A i d A l a Ser H i 3 Val G l u Hls Val L y s Ser lie P h e G l u T h r L e u T r p net Ser P h e L e u A l a A l a L e u T h r P r o P r o P h e L y s A s p T y r A S p A s p Ile GAC GTC TAT TAT GCT GCT TCC CAC GTT GAG CAT GTT AAA TCA ATT TTC GAG ACA CTA TGG ATG TCC TTT TTA GCA GCT CTA ACC CCC CCA TTT AAG GAT TAT GAT GAC ATT

1080 1090 1100

1120 A 3 p T h r T h r A s n LYS C y S L e u G l u G l y L e u L y s I l e Ser Ile L y s Ile A l a Sel T h r P h e A r q Ile A s n Aisp A l a A r q T h r Ser P h e Val G l y A l a L e u Val G i n P h e C y a GAC ACA ACC AAT AAG TGT TTA GAA GGC TTG AAA ATA TCA ATT AAA ATT GCT TCT ACT TTT AGA ATC AAT GAT GCT AGA ACC TCC TTT GTA GGT GCG TTA GTC CAA TTT TGT

1130 1140

1150 A m L e u G l n A 3 n L e u G l u G i u Ile L y s Val L y a A s n V a l A s n A l a Met Val I le L e u L e u G l u Val A l a L e u Ser G l u Gly A s n T y r L e u G I u G l y S e r T r p L y s A s p Ile AAC CTT CAA AAC CTT GAA GAA ATC A 2 4 GTT A M AAT GTC AAT GCA ATG GTA ATT CTT CTT GAA GTC GCG TTA TCA GAA GGA AAT TAC TTG GAG GGA TCG TGG AAG GAC ATT

1160 1110 1180

L e u L e u Val Val Ser G l n M e t G l u A r q L e u G l n L e u Ile Ser L y s G l y Ile A s p A x 9 A s p T h r Val Pro A s p Val A l a G l n A l a A r q V a l A l a A 3 n P r o A r q Val Ser T y r 1220

TTG CTG GTC GTG TCT CAA ATG GAA AGA CTA C M TTG ATA TCC &AA GGT ATC GAC AGA GAT ACG GTT CCA GAT GTT GCA CAA GCT CGT GTT GCA AAC CCT AGA GTT TCT TAC

1190 1200 I210

G l u Ser S e i A r g Ser A s " A s n T h r Ser P h e P h e A l p Val T r p G l y L y s L y s A l a T h r P r o T h r Glu L e u A l a G l n G l u LYS H i s Hz9 A s n G l n T h r L e u Ser P r o G l u Ile GAA TCA TCA AGA TCC AAT AAT ACA TCT TTC TTT GAT GTT TGG GGC AAG AAG GCA ACT CCC ACA GAA TTG GCT CAA GAA AAA CAC CAT AAT CAA ACC TTA TCA CCC GAA ATC

1230 1240 1250

1260 1270 1280 1290 Ser L y s P h e I l e Ser Ser Ser G l u L e u Val V a l L e u M e t ASP A 9 n lle P h e T h r L y s Ser Ser G i u L e u Ser G i y A s n A l a I l e Val A S P P h e Ile L y 3 A l a L e u T h r A l a TCT AAA TTC ATT TCC TCC AGT GAA TTA GTC GTT TTG ATG GAC AAT ATA TTT ACC AAA AGT TCC GAG TTA TCA GGT AAT GCT ATC GTT GAT TTT ATC AAA GCT TTA ACC GCT

1300 1310 1320 Val S e i L e u G l u G l u I l e G l u Ser Sex G l u A s n A i d S e x T h r P r o A r q Met P h e Ser L e u Gln L y S M e t Val A s p V a l CYS Tyr Tyr A a n M e t ASP A r q Ile L y s L e u G l u GTA TCT TTA GAA GAA ATT GAA TCA TCT GAA AAT GCT TCC ACA CCA AGA ATG TTT TCC TTG CAA AAA ATG GTC GAT GTA TGT TAC TAC AAT ATG GAT CGT ATC AAA CTA G M

1 3 3 0

T r p T h r P r o L e u T r p A l a Val M e t G l y L y a A l a P h e A s " L y 3 Ile A l a T h r Aign Ser AS" L e u A l a Val Val P h e P h e A l a Ile A 3 p Ser L e u A r q G l n L e u Ser M e t A r g TGG ACG CCG CTT TGG GCT GTT ATG GGA AAA GCT TTC AAC AAG ATT GCT ACA AAC TCT AAC TTA GCA GTA GTA TTT TTC GCT ATC GAT TCC CTG CGT CAA TTG TCT ATG AGA

1340 1350 1360

P h e L e u 1310

TTT TTA

G l u C y 3 GAG TGT

TTA AAG Le" Ly3

AAA AAT L y s As"

A 3 p Iie G l u G l u L e u Ser G l y P h e Glu P h e G l n H I S A 9 p P h e L e u LYJ P r o P h e G l u T y r T h r Val G l n As" Ser G l y A 3 n T h r G l u V a l G l o G l u M e t 11e 11e GAT ATT GAG GAA TTA TCA GGT TTT G A A TTT CAA CAT GAT TTT TTA A M CCT TTT GAA TAC ACG GTT CAA AAT AGT GGC AAC ACT GAA GTT CAA GAA ATG ATT ATT

1380 1390 1400

1410 1420 1430 1 4 4 0 P h e A r q A s " P h e Ile L e u T h r L y s S e r G l u Ser Ile L y s Ser G l y T r p L y 3 P r o Ile L e u G l u Ser L e u Gln T y r T h i A l a A r 9 S e i Ser T h r G I " Sei Ile Val TTT AGG AAC TTC ATA TTG ACA AAA TCG GAA AGT ATC AAA TCT GGC TGG AAG CCT ATT CTT GAA TCC TTA CAA TAT ACG GCT CGC TCA AGC ACT GAA TCC ATT GTA

1450 1460 T h r G l n L e u L e u Val Ser A s n A s p Ile Val T h r A s " H 1 3 P h e G l u A s " Val P h e Ser G l n G l u A S P A l a P h e Ser Glu L e u Val G l y Val P h e A r g G l u Ile T h r ACG CAA TTG CTG GTT AGC RAT GAT ATT GTG ACA AAT CAC TTC GAA AAC GTA TTC TCT CAA GAA GAT GCC TTT TCT GAG TTA GTT GGT GTC TTC AGA GAA ATC ACC

1410

1490 L y 3 A x 9 P h e G l n L y s L e u Ser L e u H ~ S A l a L e u G l u Sei Leu A r q L y 5 Met T h r G l n A s " Val A l a A s p I l e C y 3 P h e T y r A m G l u A s n L y g T h r G l u G l u G l u AAG AGA TTC CAA AAG CTA TCT CTA CAT GCT TTG GAG TCT CTA AGA AAG ATG ACT CAG AAC GTC GCA GAC ATC TGT TTT TAC AAT GAA AAT AAG ACT GAA GAA GRI\

1500 1510

1520 A r 9 L y 3 H I S A 3 0 A S P A i d Leu L e u A r q G l y L y s A s p I l e P h e G l n A s p Val T r p P h e P r o Met L e u P h e C y 3 P h e A s n A s p T h r I l e Met T h r A l a G l u A s p L e u G l u val AGA AAA CAT AAC GAT GCT I T A C T T CGT GGG AAA GAC ATA TTC CAG GAT GTG TGG TTC CCT ATG TTA TTC TGC TTC AAT GAT ACG ATC ATG ACA GCT GAA GAC TTA GAA GTT

1530 1540 I 5 5 0

1560 A r q Ser A r q A l a L e u A s n T y r M e t P h e ASP A l a L e u Val A l a T y r G l y G l y L y s P h e A s " A 9 p A s p P h e T i p G l u L y 3 Ile C y 3 L y s L y s L e u L e u P h e P r o Ile P h e G l y

1510 1 5 8 0 1590

AGA TCA CGT GCA TTA AAC TAT ATG TTC GAT GCC CTA GTG GCA TAT GGT GGT AAA TTC AAT GAT GAT PTC TGG GAA AAG ATT TGT AAG AAG TTA CTA TTT CCT ATT TTC GGT

Val L e u Ser L y 3 H i 3 T r p G i u Val As" G l n P h e R a n Ser H i 5 A s p A s p L e u Ser Val T r p L e u Ser T h r T h r L e u lie G l n A l a L e u A r g A s " L e u Ile A l a L e u P h e Thr GTA TTA TCC AAA CAT TGG GAA GTC AAC CAA TTC AAT AGT CAT GAT GAT TTA AGT GTT TGG CTT TCG ACC ACG TTA ATT CAA GCC TTA AGG AAC TTG ATT GCC CTT TTT ACG

1600 1610 1620

1630 H i s T y r P h e G l u Ser L e u A s n A r 9 net L e u A s p G l y P h e L e u G l y L e u L e u V a l Ser C y 3 Ile C y 3 G l n G l u AS" A s p T h r Ile A l a A r q Ile G i y A r q Ser C y 3 L e u G l n CAT TAC TTT GAA TCG TTG M C AGA ATG CTC GAT GGA TTT TTG GGT CTG CTG GTA TCC TGT ATT TGT C U I GAA AAT GAC ACT ATT GCT AGA ATT GGG AGA TCC TGC CTG CAA

1640 1650 1660

1610 G l n L e u I l e L e u G l n As" V a l Ser L y s P h e A a n G l u T y r H 1 4 T r p A 5 n G l n I l e G l y A 4 p Val P h e A s p L y 4 L e u P h e A S P L e u T h r T h r A l a A s " Glu L e y P h e A s p T y r CAA TTG ATA TTG CAA AAC GTA TCT AAA TTC AAT GAG TAC CAT TGG AAT C M ATA GGT GAC GTA TTC GAT AAA TTG TTT GAT TTA ACC ACT GCT AAT GAA TTG TTC GAT TAT

1680 1690 1 1 0 0

A s p P r o L e u G l n G l n G l y A r 9 L y 9 Ser Ser V a l Ser H i s H i s G I * T h r T h r A s n A a p T h r Ser G l n H i s Ser A s p A s p A 9 p Ser As" A s p A r q A r q G l u As" A s p Ser A s n GAT CCG TTA CAG CAA GGA AGA AAA TCA TCT GTA TCT CAT CAC CAA ACA ACT AAT GAT ACC AGC C M CAC TCA GAT GAT GAT AGC AAC GAC AGA AGG GAG AAC GAT TCT AAT

1110 1120 1130

1740 1750 1160 1110 I l e Ser G l u T h r Val G l u A r g A l a H i 8 G i n G l u G l u Ser S e r G l u A s p Val G l y G l y A s p Met V a l G l u T h r L e u A s n G l y G l n T h r L y s L e u ASn AS" G l y AS" Sei Val ATC AGT GAG ACG GTC GAA AGA GCG CAT CAA GAA GAA TCC AGC GAA GAC GTT GGT GGT GAC ATG GTT GAA ACA CTC AAT GGG C M ACT AAG CTT ART AAT GGC M T TCC GTT

I180 1190 1800 I810 P r o T h r Val L y g A s p G l o L e u A s n P r o L y s P r o A l a Ser L e u Ser I l e P r o LyD L y s T h r L y a H l s M e t L y s A r g A s " G l u Ser As" G l u A s p Ile A r q A r q A 1 9 Ile A l n

CCG ACG GTA RAG GAC GAA TTG AAT CCA AAG CCC GCT AGT CTA AGT ATC CCC AAG AAA ACT AAG CAC ATG AAA CGA AAC GAA AGT M C GAA GAT ATA CGT AGG AGA ATA AAC

1820 I l e L y s As" Sex Ile Val Val L y s Cy$ V a l L e u G l n L e u L e u M e r Ile G l u L e u L e u A s n G l u L e u P h e G l u As" G l u A s p P h e A l a H i 3 C y 9 Ile P r o T y r LyS G l u A l a

ATC AAG ART TCT ATT GTT GTC AAA TGT GTT TTA CAG CTT TTG ATG ATA GAG CTG CTC AAC GAA TTA TTC GAG AAC GAA GAT TTT GCC CAC TGT ATC CCT TAC AAG GAA GCC

1830 I840

1860 Ile A r q I l e T h r A r q L e u L e u G l u L y s Ser T y z G l u P h e Ser A r g A s p P h e A 5 n G l u A 3 p T y i G l y L e u A r q T h r A r g Leu Val G l u A l a A r q Val Val ASP LYS lle P r o

A T T AGA ATT ACA AGA TTG TTG GAG AAA TCG TAC GAA TTT TCT CGT GAC TTT AAT GAA GAT TAT GGG TTA AGA ACA AGA CTA GTA GAG GCT CGT GTA GTT GAT AAA ATA CCA

1870 1880

1890 A 3 n Leu Leu L y s G l n G l u T h r Ser A l a A l a A l a V a l L e u L e u A s p Ile Met P h e G l n L e u T y r L e u A s " A s p A s p G l u L y l L y s A l a A s p L e u Ile T h r A I 9 L e u I l e T h r

AAC CTG TTG AAA CAA GAA ACG AGT GCT GCT GCA GTT CTC CTT GAC ATT ATG TTT CAG TTG TAT CTG AAC GAT GAT GAG AAG AAG GCT GAT TTA ATA ACT CGT CTG ATC ACC

1900 1910 1920


89 . . . D O E G E D G D E D E D E D E D E D E D

io9 N G D E D O E D V D S S S S E T S S E D

129 G E D S E S V S G E S T E S S S G E D E

149 E S D E S D G N T S N S S S G D E S G S

1 69 E E E E E E E E E E E E E E N A G E P S

1 a9 I A H Q D S V P T N D S T A P R S T H T

209 R N I S L . . .

Asp (D): 18.4

Glu (E): 28.8

Ser (S): 20.8

FIG. 5. Highly charged region of Sec7p. The predicted amino acid sequence of Sec7p from residues 89 to 213 is highlighted. The single-letter code for amino acids is used. Content of aspartate, glutamate, and serine in this highly charged region is summarized below.

14 consecutive glutamates and 13 consecutive acidic residues. Only 2 basic residues were noted in this region, and none were found in the segment from positions 87 to 204. In contrast, the remaining Sec7p contained 14% acidic and 11% basic amino acids, giving a predicted PI of 6.0. Also somewhat unusually, the acidic domain was comprised of 21% serine, compared to 9% in the remainder of the protein.

Several other proteins with highly acidic domains have been described. Nucleoplasmin, nucleolin, high mobility group 1 (HMG-l), and CENP-B (centromere protein-B) (Refs. 40-43; also see Ref. 44 for a review) are examples of nuclear proteins that share an acidic domain. Structural roles have been proposed for these domains. Indeed, polyglutamic acid can act as a template for core histone organization and regulate nucleo- some assembly in vitro (45). Although Sec7p is apparently not a nuclear localized p r ~ t e i n , ~ highly acidic domains may be structural motifs for cytosolic proteins as well. This domain, located near the amino terminus of the 230-kDa polypeptide, may be anchored to or function in the recognition of proteins or lipids on the cytoplasmic surface of the Golgi apparatus.

Given the predicted size of Sec7p, the polypeptide could serve as a template for the assembly of proteins and lipids into a "transport complex" required for traffic between compartments of the secretory pathway (3). Sec7p could alterna- tively serve as a cytostructural protein to enclose budding transport vesicles in a lattice structure reminiscent of clathrin coats. The polyacidic domain of Sec7p may play some essential role in the interaction of Sec7p with other components of intracellular protein traffic.

Acknowledgments-We thank Gustav Ammerer for his assistance with the yeast cDNA expression library. We thank Drs. Peter Bohni, Ray Deshaies, Chris Kaiser, Francois Kepes, and Jonathan Rothblatt

A. Franzusoff and R. Schekman, manuscript in preparation.

for critical reading of the manuscript. We also thank Peggy Mc- Cutchan-Smith for help in preparing the manuscript.

REFERENCES 1. Walter, P., and Blobel, G. (1980) Proc. Natl. Acad. Sci. U. S. A. 80,4412-

2. Waters, M. G., Chirico, W. J., and Blobel, G. (1986) J. Cell Biol. 1 0 3 ,

3. Balch, W. E., Dunphy, W. G., Braell, W. A., and Rotbman, J. E. (1984)

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