Strains and Plasmids
Strains and plasmids used in this study are listed in Table and Table , respectively, and their construction is described below
YPD is a complex medium with 1% Bacto yeast extract (Difco, Detroit, MI) and 2% Bacto peptone (Difco) supplemented with glucose (2%, unless noted otherwise). YPGal is the same as YPD except that it contains galactose (2%, unless noted otherwise) instead of glucose. MVCA medium consists of 0.67% yeast nitrogen base without amino acids (Difco), 0.5% vitamin assay casamino acid (Difco), and 5% carbon source. MV-lowS medium contains 0.67% yeast nitrogen base without amino acids and ammonium sulfate (Difco), 0.1 mM (NH4
, and 5% carbon source. MV-noS medium lacks (NH4
from MV-lowS medium. Nutrients corresponding to auxotrophic markers were supplemented to the MVCA, MV-lowS, and MV-noS media. Synthetic complete (SC) medium contains 0.67% yeast nitrogen base without amino acids and 2% glucose as a carbon source (unless noted otherwise) as well as various nutrients (Sherman, 1991
). SC dropout medium lacks one or two nutrients from SC medium.
Purification and Amino Acid Sequencing of Sec24p
The Sec23p/Sec24p complex was purified as described (Hicke et al., 1992
), precipitated with 5% trichloroacetate (TCA), denatured in SDS-PAGE sample buffer, and separated by 7.5% SDS-PAGE. After transfer to a nitrocellulose membrane, proteins were stained with Ponceau S. The protein band of Sec24p (105 kDa) was excised, destained with Tris-buffered saline, and washed thoroughly with distilled water. Sec24p bound to nitrocellulose was digested with trypsin, and the released peptides were purified with C18
reverse-phase HPLC. Several peaks were recovered and sequenced: P1, IWQIFQ; P2, SVQ(D/F)ILATYK; P3, VGLLATTINTLLQNL; and P4, VTAQLLSCQDSTY.
Cloning of SEC24
Three sets of sense and antisense degenerate oligonucleotides were synthesized based on the amino acid sequences of tryptic peptides P1 (1 and rev-1) and P2 (2A, rev-2A, 2B, and rev-2B): 1, 5′-AT(A/T/C)TGGCA (A/G)AT(A/T/C)TT(T/C)CT-3′; rev-1, 5′-TG(A/G)AA(A/T/G)AT (T/C)TGCCA(A/G/T)AT-3′; 2A, 5′-AT(A/T/C)TT(A/G)GCNACNTA (T/C)AA-3′; rev-2A, 5′-TT(A/G)TANGTNGC(T/C)AA(A/T/G)AT-3′; 2B, 5′-AT(A/T/C)CTNGCNACNTA(T/C)AA-3′; rev-2B, 5′-TT(A/G)TANGTNGCNAG(A/T/G)AT-3′.
PCR was conducted with the primer pairs 1/rev-2A or rev-2B and rev-1/2A or 2B with the use of genomic DNA from RSY255 as a template. Thirty reaction cycles (each cycle was 0.5 min at 93°C, 1.5 min at 50°C, and 3 min at 72°C) were carried out, followed by a 5-min incubation at 72°C. A PCR product of 0.45 kilobase (kb) was obtained with primers 1 and rev-2A. This product was also detected in a similar reaction at a higher annealing temperature (53°C). This fragment was subcloned into an SmaI site of pBluescript II KS(+) (Stratagene, La Jolla, CA), giving pTYB121, and sequenced. An ORF was shown to span the entire 453-base pair (bp) insert. This insert was isolated, labeled with [α-32P]dCTP by means of the random primer DNA-labeling system (Amersham, Arlington Heights, IL), and used as a probe to screen yeast genomic libraries in YEp24 and YCp50. Screening was carried out according to the protocol provided by Amersham. We isolated four positive clones from YEp24 libraries (pTYY111–pTYY114) and three from YCp50 libraries (pTYY211–pTYY213). All seven clones shared a 0.5-kb EcoRI fragment, a 0.9-kb PstI fragment, and a 3.0-kb NcoI fragment that hybridized with the probe DNA. The EcoRI fragments from pTYY113 (3.3 and 0.5 kb) were subcloned into an EcoRI site of pBluescript II KS(+), and the NcoI fragment from pTYY212 (3.0 kb), converted to blunt ends by the Klenow enzyme, was introduced into a SmaI site of pBluescript II KS(+). These plasmids were used for sequencing.
Construction of Plasmids for Purification of YNL049C-encoded Protein (Iss1p)
A sequence coding for a stretch of six histidine residues was introduced in front of the termination codon of YNL049C (ISS1) as follows. The following four PCR primers were synthesized: TKPr1, 5′-CAGTAACCTCACTTAACCTATG-3′; TKPr2, 5′-TGTTAGTGATGGTGATGGTGATGTCTGTTGATACTAGTCTTCATAC-3′; TKPr3, 5′-ACAGACATCACCATCACCATCACTAACAATCAGTCTTTCTTTAATCTT-3′; and TKPr4, 5′-ATATGGCCATTTATCACGAATAC-3′.
His6 residues are encoded by the underlined sequence of TKPr3 and the sequence complementary to the underlined sequence of TKPr2. TKPr1 anneals to the antisense strand of the ISS1 ORF, ~650 nucleotides upstream from the termination codon. A part of TKPr2, TCTGTTGATACTAGTCTTCATAC, anneals to the sense strand of the 3′ end of ISS1 ORF. A part of TKPr3, TAACAATCAGTCTTTCTTTAATCTT, anneals to the 3′-flanking region and termination codon of ISS1. TKPr4 anneals to the 3′-flanking region of ISS1, ~900 nucleotides downstream from the termination codon. TKPr2 and TKPr3 anneal to each other. The plasmid pRH200 carries the ISS1 gene on a ClaI–XbaI genomic DNA fragment. The first-stage PCR was carried out with TKPr1 and TKPr2 and with TKPr3 and TKPr4 with the use of pRH200 as a template. We obtained an ~650-bp fragment with TKPr1 and TKPr2 and an ~900-bp fragment with TKPr3 and TKPr4. We used these two fragments as templates in the second-stage PCR, in which TKPr1 and TKPr4 were used as primers. This second-stage PCR yielded an ~1.6-kb DNA encoding the C terminus of Iss1p that included His6 residues as well as a termination codon and the 3′-flanking region of ISS1.
I fragment of pRH200 containing ISS1
was ligated with pGEM4Z (Promega, Madison, WI) digested with Acc
I and Xba
I to generate pTKB1. The 1.4-kb Acc
I region of pTKB1 corresponding to the 3′ end and 3′-flanking region of ISS1
was replaced by the 1.4-kb Acc
I fragment of the above PCR product encoding the His6
-tagged C terminus of Iss1p. This plasmid was named pTKB2. We sequenced the region of pTKB2 derived from the PCR product to ensure that no mutation had occurred as a result of PCR. Xba
dIII fragments of pTKB1 and pTKB2 were introduced into the Xba
dIII site of YEp352 (2μ URA3
) to obtain pTKY4 and pTKY6, respectively. The Pst
I fragment (3.5 kb) of pTKY6 encoding His6
-tagged Iss1p was introduced into the Pst
I site of p425GAL1 (2μ LEU2
) to express the ISS1
coding sequence from the GAL1
promoter (Mumberg et al., 1994
). This plasmid, pTKY7, was used for Iss1p purification.
dIII fragment (2.9 kb) of pTYY121 (YEp351 containing SEC23
) was introduced into p426GAL1 (2μ URA3
) digested with Spe
I (blunted) and Hin
dIII so that SEC23
could be expressed under control of the GAL1
promoter (Mumberg et al., 1994
). This plasmid was named pTKY9.
Purification of His6-tagged Iss1p
RSY620 was transformed with pTKY7 and pTKY9. The cells were grown in SC-Ura-Leu (2% glucose) to early stationary phase and used to inoculate 6 l of SC-Ura-Leu (2% raffinose) at an initial OD600 of 0.005. The cells were grown at 30°C for 1 d until an OD600 of ~1.2 was reached. At this time, 1/100 volume of 20% galactose was added (final concentration of 0.2%) and incubation continued for 5 h, to an OD600 of ~2.7, for overproduction of Iss1p and Sec23p. The cells were harvested and washed twice with distilled water. About 25 g of cells (wet weight) were obtained from a 6-l culture. The cells were stored at −80°C until use.
The frozen cells were suspended with HSLB (0.75 M potassium acetate, 50 mM HEPES, 0.1 mM EGTA, 20% [wt/vol] glycerol; final pH was adjusted to 7.0 with 5 M KOH) to a final volume of 70 ml. Immediately before cell disruption, protease inhibitors and reducing agent were added to the following final concentrations: 1.4 mM 2-mercaptoethanol, 1 μM leupeptin, 1 μM pepstatin A, 1 mM ε-aminocaproic acid, and 0.5 mM PMSF. Cells were disrupted in a bead-beater chamber filled with a half-volume of glass beads (0.5 mm diam, Biospec Products, Bartlesville, OK) by five 1-min periods of agitation with 2-min intervals for chilling. The lysate was recovered, and the glass beads were washed once with 20 ml of HSLB supplemented with the protease inhibitors and 2-mercaptoethanol. The total lysate (80 ml) was centrifuged at 13,000 × g for 10 min, and the resulting supernatant was centrifuged at 40,000 rpm (~186,000 × g) for 75 min (45Ti rotor, Beckman, Palo Alto, CA) to obtain a high-speed supernatant fraction.
The supernatant (46 ml) was loaded onto a 10-ml nickel-nitriloacetic acid (Ni-NTA) agarose column (Qiagen, Valencia, CA) equilibrated with HSLB with the protease inhibitors and 2-mercaptoethanol. The column was washed successively with 90 ml of B-II [0.75 M potassium acetate, 50 mM 2-(N-morpholino)ethanesulfonic acid, 0.1 mM EGTA, 20% (wt/vol) glycerol, 40 mM imidazole (pH 6.3 adjusted with 5 M KOH)], 20 ml of B-III [0.75 M potassium acetate, 50 mM HEPES, 0.1 mM EGTA, 0.25 M sorbitol, 20% (wt/vol) glycerol, 40 mM imidazole (pH 7.0)], 35 ml of B-IV100 (same as B-III except 100 mM imidazole), and finally 35 ml of B-IV200 (same as B-III except 200 mM imidazole). B-II, B-III, and B-IV100 contained the protease inhibitors. Sec23p/Iss1p was eluted from the column with B-IV500 (same as B-III except 500 mM imidazole). In a typical preparation, 1.5 mg of Sec23p/Iss1p was obtained from 25 g of cells (wet weight). Fractions that contained Sec23p/Iss1p were frozen in liquid nitrogen and stored at −80°C.
Disruption of SEC24 and ISS1
pTYB131 is a pBluescript II SK(+) derivative harboring the 4.2-kb Xho
dIII fragment from pTYY113 with the entire SEC24
gene. The Bgl
I fragment (1.6 kb) of pTYB131 was replaced by the 2.5-kb Bgl
I fragment of YEp13 containing the LEU2
gene to yield pTYY303. A 3.7-kb Bam
dIII fragment from pTYY303 containing a partial SEC24
disrupted by LEU2
was introduced into the diploid strain RSY612 to disrupt one of the chromosomal copies of SEC24
. The resulting heterozygous disruption was named RSY866. We confirmed the disruption by Southern blot analysis. Tetrad analysis was performed as described (Sherman et al., 1983
We deleted one of the chromosomal copies of ISS1
in the diploid strain YPH501 (Sikorski and Hieter, 1989
) as follows. Two PCR primers (TKPr12, 5′-CCTTCTTCCATTAATGATCGACAGCTGCAGTGAATAGCAGATTGTACTGAGAGTGCACC
-3′; and TKPr13, 5′-GGTTAATAAAGATAAAGATTAAAGAAAGACTGATTGGCATAT-GATCCGTCGAGTTCAA
-3′) were used to amplify the HIS3
gene on pRS313 (Sikorski and Hieter, 1989
). The underlined sequences of TKPr12 and TKPr13 anneal to the 5′ and 3′ regions of HIS3
, respectively. YPH501 was transformed with the amplified DNA fragment, and His+
transformants were selected. The transformants were sporulated and dissected to obtain a haploid cell with a disruption of the ISS1
A second disruption (iss1-Δ2::TRP1) that replaced amino acids 116–622 of ISS1 with the TRP1 marker was made by one-step disruption of the chromosomal ISS1 gene. The disruption plasmid (pRH247) was constructed as follows. A SpeI fragment of pRH200 was cloned into pRS306, creating pRH217. After deletion of the EcoRI site from the polylinker, the 1.5-kb BglII–EcoRI fragment of pRH217 was replaced with a 1-kb fragment containing the TRP1 marker, creating pRH247.
A trp1 diploid, CKY19, was transformed with the 2.3-kb SpeI fragment of pRH247, yielding CKY498. Tetrad analysis of CKY498 gave 2:2 segregation of TRP1. Integration of TRP1 at the ISS1 locus was confirmed by Southern blotting
Construction of Yeast Strains for the Galactose Shut-Off Experiment
The SEC24 ORF was fused to the GAL1 promotor as follows. A 5′-terminal region of SEC24 was amplified and mutated by PCR to introduce an XbaI site in front of the initiation codon. No misincorporation was found by sequencing. The amplified fragment was subcloned into the HincII site of pBluescript II SK(+) to obtain pTYB133. We replaced the BamHI–SacI region of pTYB133 with a 3.0-kb BamHI–SacI fragment from pTYB131 to obtain the complete ORF (pTYB134). A 3.1-kb XbaI–HindIII fragment of pTYB134 was isolated and introduced downstream of the GAL1 promoter on pBM743 followed by a multicloning site. The resulting plasmid, pTYY214, was introduced into RSY866. After sporulation of the transformant, a haploid segregant was obtained in which SEC24 was expressed under the control of the GAL1 promoter. RSY875 is leu2-3,-112 his3-12,15 trp1-1 ura3-1 ade2-1 GAL2 sec24::LEU2 (pTYY214 [URA3 Gal1p–SEC24]).
Multicopy Suppression Analysis
The XhoI–HindIII fragment (4.2 kb) from pTYB131containing SEC24 was ligated into YEp352 (2μ URA3) digested with SalI and HindIII to obtain pTYY115. The HindIII fragment containing SEC23 was introduced into the HindIII site of YEp352 and pTYY115, giving pTYY122 and pTYY116, respectively. Various temperature-sensitive sec mutant strains were transformed with these plasmids.
Construction of ISS1-overexpressing Yeast Strains with SEC24 Disrupted
RSY866 (MATa/α SEC24/sec24::LEU2) was transformed with pTKY4 (2μ URA3 ISS1). The resultant strain was sporulated, and the asci were dissected. Four strains derived from the same tetrad were named TKY1 (MATa SEC24 [pTKY4 (URA3 ISS1)]), TKY2 (MATα SEC24 [pTKY4 (URA3 ISS1)]), TKY3 (MATa sec24::LEU2 [pTKY4 (URA3 ISS1)]), and TKY4 (MATα sec24::LEU2 [pTKY4 (URA3 ISS1)]).
Similar strains were constructed with pTKY6 (2μ URA3 His6-tagged version of ISS1) instead of pTKY4 and named TKY5 (MATa SEC24 [pTKY6]), TKY6 (MATα SEC24 [pTKY6]), TKY7 (MATa sec24::LEU2 [pTKY6]), and TKY8 (MATα sec24::LEU2 [pTKY6]).
We constructed the following plasmid and yeast strain to regulate the ISS1 expression level in the sec24-disrupted background. The BamHI–HindIII fragment (3.5 kb) of pTKY7 containing ISS1 was introduced into p426GAL1 (2μ URA3) digested with BamHI and HindIII. The resultant plasmid (pTKY11) was introduced into the diploid strain RSY866, in which one of the SEC24 genes had been disrupted by LEU2. The transformant was sporulated and dissected on a YPGal plate to allow the expression of ISS1. We obtained a haploid Leu+ and Ura+ strain, TKY22 (sec24::LEU2 [pTKY11 (URA3 Gal1p–ISS1)]), and a Ura+ strain, TKY23 (SEC24 [pTKY11 (URA3 Gal1p–ISS1)]).
For pulse-chase analysis of carboxypeptidase Y (CPY) during Sec24p depletion, RSY875 was grown in MV-lowS (galactose) with appropriate nutrients and then transferred to fresh MV-lowS (galactose) or MV-lowS (glucose) with the supplements. After 9, 12, and 15 h of incubation, 3.0-OD600
-unit cells were collected, washed, and transferred to 5 ml of MV-noS (galactose) or MV-noS (glucose). Cells were labeled with 9.3 MBq Trans35
S-label (ICN, Costa Mesa, CA) for 10 min and then chased for 60 min. Aliquots (1.2 OD600
units of cells) were withdrawn before and after the chase, and lysates were prepared with glass beads as described (Rothblatt and Schekman, 1989
). Radioactive proteins immunoprecipitated with anti-CPY antibody were separated on SDS-PAGE and detected with a PhosphorImager (Molecular Dynamics, Sunnyvale, CA).
We conducted a pulse-chase experiment for the iss1 null strain and the ISS1-overexpressing, sec24-disrupted strain as follows. The cells were grown in SC dropout medium to late log phase and then transferred to fresh SC dropout medium (initial OD600 = 0.15). After a 2.5-h incubation at 30°C, the cells were collected, washed three times with SC-Met dropout medium, and suspended in SC-Met dropout medium (OD600 = 0.3). After a 15-min incubation at 30°C, 35S-Promix (Amersham) was added (1.5 MBq for 0.3-OD600-unit cells). After a 7-min incubation at 30°C, methionine and cysteine were added (final concentration of each amino acid was 0.6 mg/ml) and incubation was continued at 30°C. Cells (0.3 OD600 unit) were taken from the solution 0, 5, 15, 30, and 60 min after the addition of methionine and cysteine. The cell suspension was mixed with an equal volume of 10 mM NaN3/10 mM NaF on ice, collected by centrifugation, and washed once with 10 mM NaN3/10 mM NaF. The cells were resuspended in lysis buffer (1% SDS, 50 mM Tris-HCl, pH 7.5, 1 mM EDTA, 1 mM PMSF) (100 μl for 0.3-OD600-unit cells) and disrupted with glass beads. Radioactive proteins immunoprecipitated with anti-CPY antibody or anti-Gas1p antibody were separated by SDS-PAGE and detected with a PhosphorImager (Molecular Dynamics).
RSY875 grown in YPGal (5%) to OD600
= 0.15–0.6 was collected and suspended in sterile distilled water to OD600
= 6. Fifty milliliters of YPD (5%) and YPGal (5%) were inoculated with 0.1 ml of the RSY875 suspension. After a 9-h incubation at 30°C, the cells were fixed with glutaraldehyde followed by potassium permanganate, as described by Kaiser and Schekman (1990)
. Briefly, 50% glutaraldehyde was added to cultures (final concentration, 1%) for 10 min. Then the cells were centrifuged, washed, and resuspended in 4% KMnO4
for 2–4 h at 4°C. Fixed cells were collected, washed several times in water, and incubated in 2% uranyl acetate for 12–16 h at 4°C. After several rinses in water, the samples were dehydrated in an ethanol series and embedded in Spurr's medium. Thin sections were stained with lead citrate and viewed in a JEOL
100 electron microscope (JEOL
, Tokyo, Japan).
α-Factor Halo Assay
A Δsst2 strain, whose growth is arrested in the presence of α-factor, was grown in YPD at 30°C to exponential phase and suspended in YPD containing 1% agar to a final OD600 of 3 × 10−4. A YPD plate was overlaid with this suspension. To examine α-factor secretion, strains were grown to stationary phase in SC dropout medium and spotted (0.01 OD600 unit/spot) on the Δsst2-covered plate. The plate was incubated at 30°C for 2 d.
In Vitro ER Vesicle-budding Assay
GPY60 was grown at 30°C in YPD to exponential phase, and microsomes were prepared as described (Wuestehube and Schekman, 1992
). Purified microsomes were adjusted to 40 OD280
(~8 mg protein/ml) in B88 (20 mM HEPES, pH 6.8, 250 mM sorbitol, 150 mM potassium acetate, 5 mM magnesium acetate). The preparation was frozen in liquid nitrogen and stored at −80°C.
The microsome-based α-factor packaging assay was carried out as follows based on the method described (Baker et al., 1988
; Rexach and Schekman, 1991
; Kuehn et al., 1996
S]Prepro-α-factor was posttranslationally translocated into microsomes in the presence of 1× ATP regeneration mix (Baker et al., 1988
) at 10°C for 30 min. Microsomes (400 μg of protein) containing [35
S]gpαF were washed once with 1 ml of B88, resuspended in 50 μl of B88, mixed with 50 μl of B88 containing 4.2 M urea (final concentration, 2.1 M), and incubated at 0°C for 10 min. After addition of 1 ml of B88, microsomes were collected by centrifugation and washed twice with 1 ml of B88. Budding reactions were carried out in 50 μl of B88 containing 20 μg of urea-washed microsomes, 1× ATP regeneration mix, 0.1 mM GMP-PNP, and appropriate amounts of Sar1p, Sec13p/Sec31p, Sec23p/Sec24p, and Sec23p/Iss1p, whose concentrations are described in RESULTS. The mixture was incubated at 20°C for 30 min, unless noted otherwise, and chilled on ice for 5 min. Portions of the total reaction and the medium-speed supernatant (MSS) (12,000 × g
, 4 min) were collected. The amount of trypsin-resistant, concanavalin A–precipitable [35
S]gpαF in the MSS was divided by the amount in the total fraction to determine the percentage of α-factor packaged into the vesicles.
The large-scale budding reaction was carried out as follows to isolate the vesicles derived from the ER. For each reaction, microsomes containing 2 mg of proteins were used. Microsomes were incubated with 1× ATP regeneration mix in 1 ml of B88 for 30 min at 10°C. After being washed once with 1 ml of B88, they were incubated with 2.1 M urea in 300 μl of B88 for 10 min at 0°C. After addition of 1 ml of B88, microsomes were collected by centrifugation and washed twice with 1 ml of B88. A budding reaction was carried out at 20°C for 30 or 60 min in 1 ml of B88 containing 2 mg of microsomes, 1× ATP regeneration mix, 0.2 mM GMP-PNP, 65 μg of Sar1p, 120 μg of Sec13p/Sec31p, and 35 μg of either Sec23p/Sec24p or Sec23p/Iss1p. After 5 min on ice, a 50-μl aliquot reaction mixture was taken as total, and the remaining solution was centrifuged (14,000 × g, 4 min) to obtain a MSS fraction. A sucrose density gradient consisting of 0.3 ml of B88 containing 70% (wt/wt) sucrose and 2.5 ml of B88 containing 15% (wt/wt) sucrose was overlaid with 750 μl of the MSS. After centrifugation at 50,000 rpm (~250,000 × g) for 2 h (SW55, Beckman), the interface (~0.5 ml) between 15 and 70% sucrose was collected, and its sucrose concentration was adjusted to 55% (wt/wt) with the use of B88 containing 70% (wt/wt) sucrose. The final volume was ~0.8 ml, and 0.55 ml of this solution was placed on the bottom of a sucrose density gradient consisting of B88 containing 52.5, 50, 45, 40, 35, and 25% (wt/wt) sucrose (from the bottom to the top). The volume of each of the bottom three layers was 0.5 ml, and the volume of each of the top three layers was 1 ml. This gradient was centrifuged at 50,000 rpm (~250,000 × g) for 20 h (SW55, Beckman), and fractions (0.4 ml × 13) were collected from the top with a density gradient fractionator (ISCO, Lincoln, NE). Proteins in these fractions were concentrated by TCA precipitation.
Sarlp, Sec13p/Sec31p, and Sec23p/Sec24p were purified as described previously (Barlowe et al., 1994
; Yeung et al., 1995
; Salama et al., 1997
). DNA manipulation was done according to Sambrook et al. (1989)
. Yeast cells were transformed by the lithium acetate method (Ito et al., 1983
). Protein concentrations were determined with the Bio-Rad
(Richmond, CA) protein assay kit with the use of BSA as a standard. Silver staining was carried out as described (Bloom et al., 1987
). Western blot analysis was performed with a nitrocellulose membrane, and ECL (Amersham) was used for detection.