Plant Cell Physiol. 39(6): 590-599 (1998)
JSPP © 1998
Isolation of a Tobacco cDNA Encoding Sari GTPase and Analysis of Its
Dominant Mutations in Vesicular Traffic Using a Yeast Complementation
System
Masaki Takeuchi 12 , Masahito Tada 1 , Chieko Saito 1 , Hideki Yashiroda1 and Akihiko Nakano 2
1
2
Department of Biological Sciences, Graduate School of Science, University of Tokyo, Hongo, Bunkyo-ku, Tokyo, 113-0033 Japan
Molecular Membrane Biology Laboratory, RIKEN (The Institute of Physical and Chemical Research), Wako, Saitama, 351-0198
Japan
The cDNA clone of NtSARl, a gene encoding the
small GTPase Sarlp which is essential for vesicle formation from the endoplasmic reticulum (ER) membrane in
yeast, has been isolated from Nicotiana tabacum BY-2
cells. NtSARl as well as AtSARl cDNA isolated from
Arabidopsis thaliana [d'Enfert et al. (1992) EMBO J. 11:
4205] could complement the lethality of the disruption
of SARI in yeast cells in a temperature-sensitive fashion.
They also suppressed yeast secl2 and secl6 temperaturesensitive mutations as yeast SARI does. Using this complementation system, we analyzed the phenotypes of several mutations in plant SARI cDNAs in yeast cells. The
expression of NtSARl H74L and AtSARl N129I showed
dominant negative effect in growth over the wild-type
SARI, which was accompanied by the arrest of ER-toGolgi transport. Such dominant mutations will be useful to
analyze the role of membrane trafficking in plant cells, if
their expression can be regulated conditionally.
Key words: Arabidopsis thaliana — ER-to-Golgi protein
transport — Nicotiana tabacum BY-2 cells — Saccharomyces cerevisiae — SARI — Small GTPase.
The secretory pathway exports a variety of proteins to
the cell surface and is thus essential for expansion and elongation of cells. The formation of cell plates in plants also requires secretory activities. Since the development and morphogenesis of higher plants require strict regulation of
these processes, understanding of the secretory mechanisms is important in plant research. However, such studies
have not been extensively performed with plant cells, even
though a large body of information has accumulated in the
yeast Saccharomyces cerevisiae and mammalian cells. The
aim of this study is to construct a system to utilize our
knowledge in the yeast secretory pathway and apply to
plants by genetic means.
In eukaryotic cells, newly synthesized secretory proteins first enter the lumen of the endoplasmic reticulum and
Abbreviations: CPY, carboxypeptidase Y; GAP, glyceraldehyde-3-phosphate dehydrogenase.
are then conveyed to the cell surface via the Golgi apparatus. The transport between these secretory organelles
is mediated by small vesicles and is thus called vesicular
traffic. The transport from the ER to the Golgi apparatus
represents the first step of vesicular traffic and has been best
studied among the processes of secretion. In yeast, more
than 20 gene products have been identified as components
of the machinery required for the ER-to-Golgi transport.
They include two low-molecular-weight GTPases, Sarlp
and Yptlp (for reviews, see Pryer et al. 1992, Ferro-Novick
and Novick 1993).
Studies from our and other laboratories in the past
decade have elucidated that Sarlp is essential for the formation of transport vesicles from the ER membrane (Nakano
and Muramatsu 1989, Oka et al. 1991, Barlowe et al. 1993,
Oka and Nakano 1994). In the active, GTP-bound state,
Sarlp gathers coat proteins (COP II: Sec23p/Sec24p and
Secl3p/Sec31p complexes; Barlowe et al. 1994) onto the
ER membrane and promotes budding of vesicles. After the
formation of the COP II-coated vesicles is completed,
Sarlp executes its GTPase activity and is released as the
inactive, GDP-bound form from the vesicle membrane
together with other coat proteins. This uncoating process
exposes a set of membrane proteins and enables the targeting and fusion of the vesicles to the Golgi membrane
with the help of Yptlp. Secl2p and Sec23p play important
roles in the GTPase cycle of Sarlp by acting as the guanine
nucleotide exchange factor and the GTPase-activating protein, respectively (Nakano et al. 1988, Barlowe and
Schekman 1993, Yoshihisa et al. 1993).
In order to handle secretory processes in plants, it is
imperative to obtain plant secretory genes as a tool of analysis. Several plant homologues of SARI and YPT1 have
been isolated. AtSARl cDNA has been cloned from Arabidopsis thaliana by taking advantage of the fact that the
overexpression of the SARI gene suppresses the temperature-sensitive growth of the yeast secl2 ts mutant (d'Enfert
et al. 1992). cDNAs of tomato SAR2 (Davies 1994) and
Brassica BSARla and BSARlb (Kim et al. 1997) have been
identified by similarity. However, it was not examined
whether these plant SARI homologues in fact function as
the authentic SARI gene. In the case of YPT1, several
plant homologues were shown to complement yeast yptl
590
591
Tobacco SARI in vesicular traffic
mutants (Cheon et al. 1993, Park et al. 1994, Fabry et al.
1995, Loraine et al. 1996, Kim et al. 1996).
Our strategy to analyze the roles of protein secretion
in plants is to introduce and conditionally express dominant mutations of secretory genes in plant cells. SARI is
one of the best tools to do this because we have already
shown that many missense mutations of yeast SARI cause
a dominant negative effect in growth when they are overexpressed in the wild-type yeast cells (Nakano et al. 1994,
Yamanushi et al. 1996). As an experimental system, we
chose a tobacco suspension culture cell line, BY-2 (Nagata
et al. 1992). To construct a homologous transgenic system,
we isolated in this study the tobacco cDNA of the SARI
gene (NtSARl) by cross-hybridization screening of the BY2 cDNA library using AtSARl as a probe. Both NtSARl
and AtSARl could complement the deletion mutant of
yeast SARI. Using this complementation system, we performed detailed characterization of NtSARl and AtSARl
cDNAs especially with a focus on dominant mutations.
Materials and Methods
Plant, yeast and bacterial strains—The tobacco BY-2 cell
line derived from Nicotiana tabacum L. cv. Bright Yellow 2 was
kindly provided by T. Nagata of the University of Tokyo and
maintained as a suspension culture in a modified LS medium
(Linsmaier et al. 1965) at 27°C in the dark. The tobacco plants of
N. tabacum L. cv. Xanthi were used for preparation of tissue specific RNAs. Yeast S. cerevisiae strains used in this study are listed
in Table 1. Yeast cells were usually grown in YPD (1% Bacto yeast
extract, 2% polypeptone and 2% glucose) or MVD (0.67% yeast
nitrogen base without amino acids and 2% glucose) medium.
MCD medium is MVD containing 0.5% casamino acids. When
the expression of the GAL1 promoter is necessary, yeast cells were
cultured in MCGS (0.67% yeast nitrogen base without amino
acids, 0.5% casamino acids, 5% galactose and 0.2% sucrose).
Escherichia coli strains, DH5a (Sambrook et al. 1989) and XL1Blue (Bullock et al. 1987), were used for DNA manipulations.
E. coli strain Y1090 (Sambrook et al. 1989) was used for the preparation of lambda phage DNA.
Isolation of the NtSARl cDNA clone by cross hybridization
—A Agtl 1-based BY-2 cDNA library, which was constructed from
poly (A) + RNA of BY-2 cells on the second day of subculture, was
kindly provided by Y. Takahashi of the University of Tokyo and
used for cross-hybridization screening. As the probe, a 442-bp
DNA fragment was synthesized by PCR using the two primers
designed from the conserved regions between Arabidopsis SARI
and tomato SAR2 [5-GATAATGCTGGCAAAACTAC-3' (20mer); ACAATGCTGCACAT(G/A)AA(G/C)ACCTC-3' (23-mer)].
The Arabidopsis SARI cDNA (pCEY302; d'Enfert et al. 1992)
was used as the PCR template. After the first screening of the
library equivalent to 3 x 104 PFU, positive plaques were picked up
and subjected to the second screening. DNA sequences were determined by the dideoxynucleotide chain termination method using a
DNA sequencer Model 373A (Applied Biosystems).
Plasmids and DNA manipulations—Yeast multicopy
plasmids, pYO324 and pYO326 (Ohya et al. 1991), and a singlecopy plasmid, pRS314 (Sikorski and Hieter 1989), were used in
this study. Single-copy plasmids, YCpUG-578T (pRS316 derivative) containing the GAL1 promoter and the CMK1 terminator
(obtained from H. Qadota of the University of Tokyo) and its derivative pTUl containing the GAP promoter (Ueda et al. 1996),
were also used. The GAP promoter is derived from the TDH2
gene encoding glyceraldehyde-3-phosphate dehydrogenase and is
used for strong, constitutive expression in yeast.
The Agtl 1 clone containing the N. tabacum SARI cDNA was
subcloned into pBluescript II KS + and named II-4. II-4 contained
two cDNA inserts, 1,020-bp tobacco SARI cDNA and an unrelated 360-bp fragment in the downstream. To subclone the cDNA
region that contains the SARI open reading frame (ORF), the 1.3kb Bglll-BamHl fragment of II-4 was first inserted into the Bglll
site of pTUl. This construct was digested by EcoKl to generatethe 1.0-kb EcoKl-EcoRl fragment, which was again subcloned
into the £coRI site of pTUl (pTUNSl). The GAP-promoterORF-terminater cassette was further subcloned into pRS314,
pYO324 and pYO326.
AtSARl
cDNA (1.0-kb fragment) was excised from
pCEY302 by digestion with Notl and subcloned into the £coRV
site of pBluescript II KS + . The 1.0-kb £coRI/7/mdIII fragment
from this construct was subcloned into the EcoRl-Hindlll sites of
Table 1 Yeast strains used in this study
Strain
ANS16-7A
ANY21
ANY27
MBY10-7A
MTYA1
MTYN1
TOY223
TOY224
TOY323
YSY20
Genotype
Reference
or source
MATa. seel6-2 ura3 Ieu2 trpl his3 his4
MA Ta ura3 Ieu2 trpl his3 his4 sue gal2
MA Ta sarl::URA3 ura3 Ieu2 trpl his3 his4 /pTY5 (YCp[GALl-SARl
LEU2])
MA Ta sec!2-4 ura3 Ieu2 trpl his3 his4 sue gal2
MATa sarl::LEU2 ura3 Ieu2 trpl his3 his4 /pMTG202 (YCp[GAP-AtSARl
TRPl])
MATa sarl::LEU2 ura3 Ieu2 trpl his3 his4 /pMTG203 (YCp[GAP-MS/l/?7 TRPl])
MATa sarl::HIS3 pep4::ADE2 ura3 Ieu2 trpl his3 ade2 Iys2 /pMYY3-7 (YCp[sarl-3 TRPl])
MATa sarl::HIS3 pep4::ADE2 ura3 Ieu2.trpl his3 ade2 Iys2 /pMYY3-9 (YCp[sarl-2 TRPl])
MATa sarl::LEU2 ura3 Ieu2 trpl his3 his4 /pANY2-9 (YCpSARl URA3])
MATa sarl::HIS3 pep4::ADE2 ura3 Ieu2 trpl his3 ade2 Iys2 /pMYU4-l (YCp[GALl-SARl URA3])
A
B
C
B
A
A
D
E
D
F
A, this study; B, Nakano et al. (1988); C, Nakano and Muramatsu (1989); D, Toshihiko Oka; E, Yamanushi et al. (1996); F, Yumiko
Saito.
592
Tobacco SARI in vesicular traffic
pTUl to yield pTUASl. The whole cassette containing AtSARl
was also subcloned into pRS314, pYO324 and pYO326.
Mutations in NtSARJ and A tSARl, NtSARl D29G, NtSARl
H74L, AtSARl T51A, AtSARl D70V and AtSARl N129I, were
generated by site-directed mutagenesiss and PCR. All constructions were confirmed by sequencing.
DNA manipulations including restriction enzyme digestion,
ligation, plasmid isolation and E. colt transformation were carried
out by standard methods. Yeast transformation was performed by
a lithium acetate method (Ito et al. 1983) or a lithium thiocyanate
method (Keszenman-Pereyra and Hieda 1988).
Northern blot analysis—Total RNA was prepared by disrupting plant materials frozen by liquid N2 in a mortar with the equal
volume of 4 M guanidine thiocyanate solution containing 10 mM
Tris-HCl pH 7.5, 0.5% sodium N-dodecanoylsalcosinate and \%
/?-mercaptoethanol and phenol/chloroform/isoamylalcohol (25 :
24 : 1). The phenol/chloroform/isoamylalcohol extraction was
repeated several times.
RNA samples (15 fig RNA per lane) were separated by formaldehyde-agarose gel electrophoresis, and transferred to a nylon
membrane with 20xSSPE at room temperature for 20 h. RNA
was fixed on the membrane by UV light irradiation. Hybridization
was performed with the 1,020-bp EcoKl-EcoRl fragment of the
NtSARl cDNA as a probe at 42°C for 20 h in 5 x SSPE/50%
formamide/5 x Denhardt's solution/0.5% SDS. The membrane
was then washed twice with 2 x SSPE/0.1% SDS at room temperature for lOmin, twice with 1 xSSPE/0.1% SDS at 65°C for 20
min, and once with 0.1 x SSPE/0.1% SDS at 65°C for 20min.
The images of autoradiograms were visualized using an Imaging
Plate Scanner BAS2500 (Fuji Film).
Genomic Southern blot analysis—Genomic DNA was prepared from BY-2 cells according to the method of Murray and
Thompson (1980). Ten |ig DNA was digested overnight with appropriate restriction enzymes, separated by agarose gel electrophoresis, and then transferred onto a nylon membrane. The 1,020-bp
EcoRl-EcoRl fragment of the NtSARl cDNA was used as the
full-length cDNA probe and a PCR-amplified 245-bp DNA fragment containing the region between the stop codon and poly (A)
was used as the 3'-noncoding region probe. Hybridization was
done in 0.5 M sodium phosphate (pH 7.4), 1 mM EDTA, \% BSA
and 1% SDS, and the membrane was first washed under a low
stringency condition (twice with 2 x S S C / 0 . 1 % SDS at 65°C for
30 min each). After an appropriate exposure for autoradiography,
the membrane was further washed under a high stringency condition (twice with 0.1 xSSC/0.1% SDS at 65°C for 30 min each)
and subjected to autoradiography again.
Pulse-chase experiments—Yeast cells were first cultured overnight at 30°C in MVD medium containing 100 fM. (NH4)2SO4.
Aliquots of cells (1.2 x 108 each) were harvested, washed twice
with sterile water, resuspended in MVGS medium containing 100
/iM (NH4)2SO4, and further incubated at 30°C for 4 h and 15
min. Then, the cells were washed twice with sterile water and resuspended in 1.2 ml of MVD with no sulfate. After preincubation for
30 min at 30°C, the cells were pulse-labeled with 17.5 //Ci/107 cells
of Tran35S-label (ICN Radiochemicals) for 5 min and then chased
by the addition of 4.8 ml of 30 mM (NH4)2SO4> 2.5 mM cysteine
and 2.7 mM methionine. One-ml aliquots were withdrawn after 0,
5, 15, 30 and 60 min incubation at 30°C and mixed with 110/^1 of
100 mM NaN3. Preparation of cell extracts and immunoprecipitation with an anti-carboxypeptidase Y (CPY) antiserum were
performed according to the method of Nishikawa and Nakano
(1991). Immunoprecipitates were analyzed by SDS-PAGE and
fluorography. The images of fluorograms were visualized with
BAS2500.
Results
Cloning of the NtSARl cDNA—We had obtained the
Arabidopsis AtSARl cDNA (d'Enfert et al. 1992) as a kind
gift from C. d'Enfert (now in Pasteur Institute). To construct a homologous system to work with the tobacco BY-2
cells, we decided to clone the tobacco counterpart of
SARI. An AtSARl cDNA fragment containing the regions
conserved between Arabidopsis SARI and tomato SAR2
(Davies 1994) cDNAs was amplified by PCR and used as a
probe for hybridization screening of a BY-2 cDNA library.
Two positive plaques obtained in the first screening were
subjected to the second screening for purification. By the sequence analysis of five independent positive plaques obtained in the second round, only one cDNA clone was revealed.
This clone contained two EcoRl fragments in tandem. The
longer fragment (1,020 bp) in the upstream contained a
582-bp ORF that encodes a tobacco homologue of the
SARI family and a poly (A) stretch (Fig. 1). We suggest
that these two inserts were ligated by an accident during the
construction of the library and the longer fragment is the
true cDNA for the tobacco SARI gene, because the second
shorter EcoRl fragment in the downstream did not show
any effect in the complementation of the yeast Asarl mutant (see below), and such a sequence was not present in the
cDNA isolate of Arabidopsis SARI. We designated the
cDNA clone in the upstream NtSARl. The sequence of this
1,020-bp cDNA has been registered in databases (DDBJ,
GenBank and EMBL) with the accession number D87821.
There are three more ATG codons upstream of the
NtSARl ORF in this cDNA. However, because they are
not in frame with the ORF and were not required for the
complementation of yeast mutants, we concluded that the
fourth ATG is used for the initiation of translation. The
cDNA also contains a polyadenylation signal (AATTTA)
at 189 nucleotides downstream from the stop codon (TAG)
of the NtSARl ORF.
Divergence of the SARI gene family—The amino acid
sequence deduced from the NtSARl ORF (NtSarlp) and
its comparison with other members of the Sari family are
shown in Figure 2. The sequence identity at the amino acid
level is 93%, 88% and 62% to Arabidopsis Sarlp, tomato
Sar2p and yeast Sarlp, respectively. Basically, the consensus sequences for GTP binding (GLDNAGK, residues 2733; DLGG, residues 70-73; and NKID, residues 129-132)
and the effector domain (LVQHQPTQYP, residues 45-54)
are very well conserved. The N- and C-terminal regions are
less conserved and the evolutionary divergence of three
groups, animal, plant and fungal Sari proteins, is also
noticeable.
To investigate how many SARI genes exist in the
genome of tobacco, a Southern blot analysis was carried
Tobacco SARI in vesicular traffic
593
U I 111111 11IUIATAGAAATGGTATGATATAACACGTGATGACGTGAACAATAGAAT
60
CGACGCATTAATAAGCATTAGGGTAGATCCCAGATCTGGTGGAAGAAGAACATTACAAa
120
AGAAaGAGATCCCCCCATCGGCGATCAACGGAAAAGATGTTTCraGGGATTGGTTCTA
M F L W D W F Y
TGGCGTTTTGTCTTCTCTAGGTTTATGGCAAAAGGAAGaAAGATCaCrTTCTCGGTCT
G V L S S L G L W Q K E A K I L F L G L
CGACMTGCCGGCAAAACCACTCTTCTTCACATGCTCAAAGACGAGAGATTAGTTCAGa
D N A G K T T L L H M L K D E R L V Q H
TCAGCCGACGCAGTATCCGACATCGGAAGAGCTGAGTATCGGAAAAATCAAATTCAAAGC
Q P T Q Y P T S E E L S I G K I K F K A
GTTTGATTTAGGAGGTCATCAGATCGCTCGCCGTGTCTGGAAAGATTATTATGCCMGGT
F D L G G H Q I A R R V W K D Y Y A K V
TGATGCrGTTGTATACCTGGTAGATGCATATGACAAAGAGCGGTTTGCAGAATCAAAGAA
D A V V Y L V D A Y D K E R F A E S K K
AGAGTTAGACGCAaGCTATCTGATGAGGCCCTAGaAaGTCCCGTCCTCATTTTGGG
E L D A L L S D E A L A T V P F L I L G
AAACAAGATAGACATACCGTATGCTGCCTCGGAAGATGAGCTGCGTTATCACCTTGGTn"
N K I D I P Y A A S E D E L R Y H L G L
GAaGGTGTCAaACTGGCAAGGGAAAGGTGAGTGTTGCTGATTaAGTGTTCGTCCGT
T G V T T G K G K V S V A D S S V R P L
GGAGGTCrTTATGTGCAGTATTGTACGCAAAATGGGGTATGGTGATGGCTTTAAGTGGGT
E V F M C S I V R K M G Y G D G F K W V
TTa(^TATATAMCTA(TrTTACGCAACCA(^(rrT(^AACAGATTGTGTCCTCGTTTA
S Q Y I K
TGCTTTTCGTACAACrAI 1111 ICCCGTaGGAAAAGATGGAACCTGTTCATTTGTTCTC
180
240
300
360
420
480
540
600
660
720
780
840
AAACT<nTGAGGTCTTTTACTACrTATMTTTCTATGCGTTGAACATCTACTTAGTGTGA
900
TGAGMGCTATnAnGTTGCCCTGTAAAATTTACCTAAGGCTGGAACATTGTCAAAACT
960
CCATATTATTATTCCATTTCTTAGTTAAAAAAAAAAAAAAAAAAAA
1006
Fig. 1 Nucleotide and deduced amino acid sequences of NtSARl. NtSARl ORF (582 bp) is present at nucleotides 158 to 739. A putative poly (A) addition signal exists at nucleotides 929 to 934 and is underlined. The £coRI linker sequences on the both ends in the
original isolate are omitted in this figure.
out with the BY-2 genomic DNA digested with BamHl,
(Fig. 4). A 1.0-kb message was observed. The signal was
EcoRl and Hindlll (Fig. 3). When the full-length cDNA of much stronger on the 3rd day than on the 7th day, sugNtSARl was used as a probe, 4 to 6 bands were hybridized
gesting that the expression of NtSARl is higher during the
in a low-stringency condition and 3 to 4 bands were detect- logarithmic growth phase than in the stationary phase. To
ed in a high-stringency condition (panels A and B). With examine the expression pattern of NtSARl in tobacco
the 3'-noncoding region as a probe, in contrast, only two plant, RNA was extracted from roots, stems, leaves and
bands were detected in the low-stringency condition, one of flowers from N. tabacum and similarly analyzed by Norwhich faded further by the high-stringency wash (panels C them blotting. As also shown in Figure 4, the 1.0-kb tranand D). Since the evolutionary pressure would have con- script was observed for all of these organs indicating that
served the ORF sequence better than non-coding regions, the NtSARl gene is ubiquitously expressed in the tobacco
the 3'-noncoding probe was probably hybridizing with the organism. Essentially the same results were obtained when
most similar copies of SARI genes in the tobacco genome. the 3'-noncoding region was used as a probe (data not
In other words, the NtSARl gene exists at one or two loci shown).
in the genome of tobacco BY-2 cells and homologous but
Suppression of yeast sec!2 and secl6 ts mutants by
more distantly related genes are present as well.
plant SARI—Yeast SARI has been shown to suppress two
Expression of NtSARl in BY-2 cells and in tobacco temperature-sensitive (ts) yeast mutations, secl2 and sec!6,
plant—Total RNA was prepared from BY-2 cells on the when its gene dosage is raised (Nakano and Muramatsu
3rd and 7th days of subculture and subjected to Northern
1989). The Arabidopsis AtSARl cDNA was in fact isolated
blotting with the full-length NtSARl cDNA as a probe taking advantage of its ability to suppress the yeast sec!2
594
Tobacco SARI in vesicular traffic
GXXXXGK
ChSARla
MmSARl
CeSARl
AtSARl
BcSARla
NtSARl
NpSARl
LeSAR2
AnSARl
SpSARl
ScSARl
Consensus
M-SF-IFDWI
j^-SF-IFEWI
ChSARla
MmSARl
CeSARl
AtSARl
BcSARla
NtSARl
NpSARl
LeSAR2
AnSARl
SpSARl
ScSARl
Consensus
|RLGQHVpiLH
WLGQHVRTLH
JRIAQHVPTLH
JRLVQHQPirQH
|R!LVQHQPJQH
Mi-SF-LWDWF
M--F-LFDWF
M--F-VIDWF
|M--F-LWDWF
MI--F-LWD|(F
YSGFSSVLQF
YNGFSSVLQF
NGVtNM
Y
GICAS
Y
GVEAS
Y
GVLSS
Y
GVliSS
Y
GVLAS
Y
DLEAS
Y
DAtfAM
R
DVtfAS
•«--W-LIN|iF
MJ--F-IINWF
MAGWDIFGWF
M
.3
M--F-L.DWF Y
effector
L'GLYKKTGKL VFCGLDNAGK TTLCHMLKDD
L G L ' Y K K S G K L VFLGLDNAGK TTLLQMLKDD
LGL'ANKKGKL VFLG'CDNAG"K.*TTL'LHMLKOD
LGLWQKEAKI LFLGLDNAGK'TTLLHMLKDE
LGl!WQKEAKI LF'CGLDNXGK TTttHMLKDE
LGLWQKEAKI LFLGLi3NA'GKtlTTUHMLi<DE
LGLWQKEAKI L F L 1 ; L * D N A G K £ T T L L H M L K D E
LGtWQKDAk'I L FL'GCO'NAGK ; T f L I H M C K O E
LGL'LNKHAKL LELGEDNXGk'-tTLtHMltNO
llGGVNKHAKM Lf tGtDNXGK. JTlL'HML'KND
LGCWNKHGKL LFLGCDNAGK' TTLLHML'KND
GVtrAS LGLW.K.AK".
JW56JP* i
LFLGLONAGKTTLLHMLKDD
DXXG
MTf-TTFDL'GCHIQARRVWKN
MTFTTFDLGG HEQARRVWKN
ISFfTTYDtGG HAQARRVWKD
IKFKAFOL'GG^HQIARRVWKD
IKF.KAFDL'GG^HQIARRVWKD
Q I ARRVWKD
QIARRVttKD
IK^KAFDuVG'lHQIARRVttKD
v f
IKFKAFDL G G* HQIARRVWRD
NRFTTFDt'GG": HQQARRLWKO
VRtjTTFDL.GG ;HQQARRLWRO
IKFiTTFOLGGS-HIQiCRRLIIKKD
YLPAINGIVF
,Y|LPAINGIVF
XFPAVDAVVF
Y4YAKVDAVVY
YYAKVDAVVY
Y,YAKVDAVVY
YYAKVDAVVY
TYAKVDAVVY
Y/PEVSGIVF
YFPEVNGIVY
YFPEVNGIVF
Y^.P.VDAWY
98
98
94
93
93
93
93
93
93
93
96
100
PEAISEERLR
TDAl'SEEKLR
PG;A'LS'EDQLK
PY'A'ASEDELR
PYA'ASEDELR
PYA'ASEDELR
PYA'ASEDELR
PYAASEDELR
PD"AVSEDDVR
PGA'ISEDELK
PNAVSEAELR
H W
.|sK.ELDAL.L.DE.LA.^FLlS!GiNKI^. P.A.SEDELR
148
148
144
143
143
143
143
143
143
143
146
150
BKGSVSLKEL
IGKGNVTLKEL
GKGDVSRNEM
GKGKVTLGDS
GKGKVDLVGS
GKGKVSVADS
GKGKANLADS
GKGNINLAGT
198
198
193
193
193
193
193
193
189
190
190
JRLATLQPTJWH
PTSEELTIAG
PTSEELTIAG
PTSEQMSLGG
PTSEELSIGK
PTSEELSIGK
PTSEELSIGK
PTSEELSIGK
PTSEELSIGN
PTSEELAIGN
PTSEELAIGN
PTSEELAIGN
iBL.QHQpJ.H
PTSEELSIG.
ChSARla
MmSARl
CeSARl
AtSARl
BcSARla
NtSARl
NpSARl
LeSARZ
AnSARl
SpSARl
ScSARl
Consensus
LVDCADHERL
LVDCADHSRL
LIDVADAERM
LVDAYDKERF
LVDAYDKERF
LVDAYDKERF
LVDAFDKERF
LVDANORERF
L'VDAKDHECF
LVqCCd^FERL
LVOAADPERF
LESKEELDSlTMTDETIANVP'miL'GMKIDR
MESKVELNAL MTDETISNVP^ILILGNKIDR
QESRVELESL LQDEQIASVP jVLILGNKIDK
AESKRELDAL LSDEALATVP FLILGNKIDI
AlSKKELDAL LSDESLATVP "FLILGNKIDI
AEjSKKELDAL ISDEALATVPiFLILGNKIDI
AE'SKKELDAL 'LSDEALSTVP- FLILGNKIDI
PEAKKEtDGL LSDESLTNVP^FLJLGNKIDI
PfeSKAELDAL LAMEELAKVPjFLiL'GNKIDH
SESKAELDAL 'LAMEELARVP.;|FLILGNKIDA
DIARVEL'DAC, FNIAELKD^FVI'LGNKIDA
ChSARla
MmSARl
CeSARl
AtSARl
BcSARla
NtSARl
NpSARl
LeSAR2
AnSARl
SpSARl
ScSARl
EMFGLYGQTT
EIKGLYGQTT
WQLNIQHMCT
YHLGLTNFTT
YHLGLSNFTT
YHLGLTGVTT
YHLGLTGVTT
YHLGLTGVTT
HQLGLYQTTAALGLYQTTSALGLLNTT-
Consensus
. . L G L . . .TT g K G . V . L
flfQHQPlfc
[RLVQHQPIQY
PLVQHQgTjQY
|RVAILQP;TAH
RLAVMQPTLH
48
48
44
43
43
43
43
43
43
43
46
50
NKXD
LVD'A.K.ERF
NARPLEVFMC'ISVLKRQGYGE
NARPMEVFMC-'SVLKRQGYGE
AS RPMEVFJMCSVLQRQGYG E
GVR'PLEVFHCFSIVRKMGVGE
NVRP\EVF #
SIVRKMGYGE
SVRPLEVFHG ISIVRKMGYGD
SVRPLEV?H*C-SIVRKMGYGD
NVRPIEjflLClSllVRKMGYGE
iCKG—KVPLE GI RBI EyfRCTS VVMRQGYG E
GKG-VSKPVP GIRPJ EVFMefS VV L RQGYG E
_._._^
_^
G- — SQRIE GQRPJVE.VTFMCSVVMRNGYLE
GFRflMApiD
GFR|LSQYID
GIRlfLGQYLGFK|LSC$IN
GFKl»LS|^K
GFKl»LS|^IK
GFK|VSQ|IK
GFKt!(VSQYlK
GIRKLSQYVGFKiLAQtVAFQiLSpi
^
RJ5.EVF.MC1*SVV.R.G*YGE G F K I L S . S l .
200
Fig. 2 Comparison of Sarlp sequences from different organisms. Similar and identical amino acid residues are marked by light and
dark shadows, respectively. Three GTP-binding consensus motifs and the effector domain are overlined. Ch, Chinese hamster (CHO
cells) (559644); Mm, mouse Mus musculus (AtT-20 cells) (L20294); Ce, Caenorhabditis elegans (U58748); At, Arabidopsis thaliana
(M95795); Be, Brassica campestris (U55035); Nt, Nicotiana tabacum (BY-2 cells) (D87821); Np, Nicotiana plumbaginifolia (Y08423);
Le, tomato Lycopersicon esculentum (L12051); An, Aspergillus niger (Z67742); Sp, Schizosaccharomycespombe (M95797); Sc, Saccharomyces cerevisiae (X51667). The numbers in parentheses are the database accession IDs of GenBank.
mutation (d'Enfert et al. 1992). We examined whether
NtSARl could also suppress secl2 and secl6. First, the
cDNAs of NtSARl as well as AtSARl were placed under
the GAP promoter, which drives high and constitutive expression in yeast, and introduced into secl2 and secl6 cells.
The 100-bp EcoRl-Bglll fragment of NtSARl cDNA was
deleted to remove three non-in-frame ATG codons. We
found that both NtSARl and AtSARl suppressed secl2
and seel 6 but the degree of suppression was much better by
NtSARl than by AtSARl (data not shown). Considering
Tobacco SAR] in vesicular traffic
probe
3'-noncoding region
full-length
stringency
low
high
low
high
B E H
B E H
B E H
B E H
bP
23130 —
• '^Ji •* .•"',-
,
m
9416 —
6557 —
4361 —
• *%
•
-t
m
2322—2027 ~"
Wm
^»5
E • * * ^
*
t
$•*•
(A)
(B)
(C)
Fig. 3 Genomic Southern analysis of the NtSARJ gene. Genomic DNA isolated from BY-2 cells (10 fig) was digested with BamHl,
EcoKl and Hindlll. Each DNA sample was then separated on an agarose gel and blotted onto a nylon membrane. (A) and (B), blots hybridized with the NtSARl full-length cDNA probe and washed under a low stringency condition (2 x SSC, 0.1% SDS, 65°C) (A) and a
high stringency condition (0.1 x SSC, 0.1% SDS, 65°C) (B). (C) and (D), blots hybridized with the 3'-noncoding region probe and washed under low (C) and high (D) stringency conditions. B, E and H indicate digestion by BamHl, EcoRl and Hindlll, respectively.
the possibility that noncoding regions of these cDNAs
might have affected the expression levels in yeast, we reconstructed single-copy plasmids that contain only the ORF
regions beneath the GAP promoter and transformed the
secl2 and secl6 mutant cells again. The results are shown
in Figure 5. The suppression activity of AtSARl was markedly improved and both NtSAR] and AtSARl could suppress the ts growth of secl6 up to 35°C. This result suggests
that the 5'-noncoding region of AtSARl was somehow
disturbing its expression in yeast. For the suppression of
secl2, however, AtSARl had still very low ability as compared to yeast SARI or NtSARl even with the ORF alone
construct. NtSARl suppressed secl2 at 35 and 37°C but AtSARl did so only partially at 30°C.
Complementation of yeast sari mutants by plant
SARI—The same plant SARI constructs under control of
the GAP promoter were further tested for their abilities to
complement the yeast sa/7-deletion mutant. The yeast
SARI gene is an essential gene and its disruption is a lethal
event, but the complementation test can be performed using the regulatable expression of yeast SARI by the GAL1
promoter. The GAL1 promoter induces high-level gene expression on galactose medium but almost completely shuts
off the expression on glucose. The ANY27 strain has its
chromosomal SARI gene disrupted but harbors a plasmid,
on which SARI is expressed under control of the GAL1
promoter, and thus is viable on galactose but not on glucose (Nakano and Muramatsu 1989). ANY27 cells were
transformed with the plant SARI cDNAs and incubated on
galactose and glucose plates at various temperatures. As
shown in Figure 6, ANY27 containing vector alone could
not grow on glucose at any temperatures. However, when
the ORF construct of NtSARl or AtSARl was expressed
by the GAP promoter, cells grew at 30 and 35CC. Such complementation was not observed at 37°C. Further overexpression by introducing multicopy plasmids containing the
same constructs did not significantly change the temperature profile of the growth. Thus, the two plant SARI
cDNAs complement the deletion of the yeast SARI gene,
but they do so in a temperature-sensitive fashion. The fulllength cDNA version of AtSARl did not complement the
SARI deletion even at low temperatures (data not shown).
It was possible that a very low expression of yeast
SARI from the GAL1 promoter on glucose might have
helped the complementation by plant SARI cDNAs. To
test this, the GAL1-SAR1 plasmid (harboring the URA3
marker) of YSY20 cells was forced to drop out by the use
of the 5-fluroorotic acid (Boeke et al. 1984) in the presence
of the complementing plant SARI plasmid. Even in the
complete absence of the GAL1-SAR1 plasmid, plant SARI
s§s
Tobacco SARI in vesicular traffic
BY-2
3 7
R
Plant
S L
F
NtSARl
30'C Gal
30'C Glc
35"C Glc
37'C Glc
rRNA
Fig. 4 Northern blot analysis of the NtSARJ gene. Total RNA
was prepared from BY-2 cells on the 3rd and 7th days after subculture, or from roots, stems, leaves and flowers of tobacco plants.
Each sample (15 fig) was separated on a formaldehyde-agarose ge!
and blotted onto a nylon membrane. The blot was hybridized with
the NtSARl full-length cDNA probe and washed with 0.1 x
SSPE/0. \% SDS at 65°C. The size of the NtSARl transcript is estimated to be about 1.0 kb by comparision with ribosomal RNAs.
Upper panel, autoradiogram of the Northern blot; lower panel,
photograph of the agarose gel before the blotting to show the pattern of ribosomal RNA.
ScSARI AtSARl
vector
cDNAs could sustain the growth of the scr7-deleted cells
(data not shown). Thus, yeast cells can grow, at least at low
temperatures, solely depending on plant SARI instead of
yeast SARI.
We also performed complementation tests for sari ts
strains, sarl-2 (E112K) and sarl-3 (D32G) (Yamanushi et
al. 1996), by transforming them with single- and multicopy
plasmids containing NtSARl or AtSARl under the GAP
promoter. Neither of these plant SARI cDNAs could sup-
23'C
30'C
secie sects
v vector
ScS4RO<
vector *
y/scSARI
sec16
y/ \
AtSARl/
sec12
NtSARl NtSARl*
35'C
Fig. 5 NtSARl and AtSARl suppress the temperature-sensitive
growth of yeast secl2 and secl6 ts mutants. MBY10-7A (secll)
and ANS16-7A (secl6) cells harboring pRS314 (vector),
pMTG201 (ScSARI; yeast SARI), pMTG202 (AtSARl) or
pMTG203 (NtSARl) were streaked on MCD plates and incubated
at 23, 30 or 35°C for 3-6 d.
NtSARl
Fig. 6 NtSARl and AtSARl complement the lethality of the
yeast sari deletion mutant. ANY27 cells harboring pRS314 (vector), pMTG201 (ScSARI), pMTG202 (AtSARl) and pMTG203
(NtSARl) were streaked on MCGS (Gal) and MCD (Glc) plates
and incubated at 30, 35 or 37°C for 3-4 d. Complementation of
the sari deletion mutation by plant SARI cDNAs was observed on
glucose plates.
port the growth of these ts mutants at 35 or 37°C (data not
shown). As these sari ts mutations show some dominant
nature when overproduced (Nakano et al. 1994), perhaps
the weak complementation of the SARI function by plant
SARI cDNAs did not overcome the mutant phenotypes of
yeast sari.
Analysis of mutant versions of plant SARI cDNAs
in yeast cells—We have previously reported that many
missense mutations in the yeast SARI gene cause a dominant negative effect in growth when overexpressed in yeast
cells (Nakano et al. 1994). When the mutant versions of
SARI were placed under control of the GAL1 promoter
and introduced into the wild-type yeast cells, cell growth
was arrested several hours after the medium shift from glucose to galactose. This was due to the block of the ER-toGolgi transport caused by the overproduction of mutant
Sarlp.
To test whether the corresponding mutations in the
plant SARI cDNAs also show dominant effect over
the wild-type SARI, we constructed 5 mutants: NtSARl
D29G, NtSARl H74L, AtSARl T51A, AtSARl D70V and
AtSARl N129I. As yeast host cells, we prepared the follow-
Tobacco SARI in vesicular traffic
(A) ANY21
P2
\
Nt
WT
D29G
\
/
Nt
H74L /
/
/
Glc 30°C
597
/
At
4
WT/
/ 'At
T51A
At
\ k D70V
At \
N129IN
Gal 3OC
ANY21
P1
m
p2
1
P1
ANY21
/NtSARl
m •
(B) MTYN1
Nt
H74L
P2
ANY21
/NtSAR7H74L
P1
m
Glc 30*0
5
Gal 3CTC
(C) MTYA1
Glc 30°C
At
D70V
At
WT
At
N129I
At
T51A
Gal 3f/C
Fig. 7 Expression of mutant versions of NtSARl and AtSARl
in yeast cells. ANY21 (yeast wild type), MTYN1 (NtSAR 1-dependent yeast) and MTYA1 04/S/iflV-dependent yeast) cells harboring
the plasmids that express various plant SARI cDNAs, NtSAR]
wild-type (WT), NtSARl D29G, NtSARl H74L, AtSARl wildtype (WT), AtSARl T51 A, AtSARl D70V or AtSARl N129I under the control of the GAL1 promoter were streaked on MCD
(Glc) and MCGS (Gal) plates and incubated at 30°C for 3-5 d.
ing three strains: ANY21, yeast wild type; MTYN1,
MS/l/?7-dependent cells; and MTYA1, A t SARI-depend-
15 30 60
chase(min)
Fig. 8 A pulse-chase experiment to follow the intracellular transport of carboxypeptidase Y (CPY). ANY21 (yeast wild type),
ANY2l/NtSARl
and ANY2\/NtSARl H74L cells cultured in glucose medium were washed and resuspended in galactose medium
to induce the expression of NtSARl. At 4 h and 45 min after the
induction, cells were pulse-labeled with Tran35S-label for 5 min
and chased at 30° C. Aliquots were withdrawn at the indicated
times and subjected to immunoprecipitation with the anti-CPY
antibody, p i , ER-precursor form; p2, Golgi-precursor form; m,
mature vacuolar form.
ent cells. MTYN1 and MTYA1 are both derived from
TOY323; they have the chromosomal SARI locus disrupted and harbor a single-copy plasmid containing NtSARl
and AtSARl, respectively, under the GAP promoter.
The five plant mutant SARI cDNAs as well as their
wild-type controls were placed downstream of the GAL1
promoter on a single-copy plasmid and used to transform
the yeast cells of the above three strains on glucose plates.
Table 2 Effects of the overexpression of mutant plant SARI cDNAs on the growth of
yeast cells
Expressed
plant SARI
NtSARl
NtSARl
NtSARl
AtSARl
AtSARl
AtSARl
AtSARl
Recipient yeast
Wild type
Asarl/NtSARl
01
nt
D29G
H74L
T51A
D70V
N129I
—, no effect; + , dominant negative effect; nt, not tested.
Asarl/AtSARl
lit
nt
nt
nt
nt
598
Tobacco SARI in vesicular traffic
The growth of the transformants was examined on glucose
and galactose plates. As shown in Figure 7, the expression
of NtSARl H74L and AtSARl N129I showed dominant
negative effects in growth to the respective wild-type SARI
(MTYN1 and MTYA1) and even to the yeast wild-type
SARI (ANY21). AtSARl T51A also showed a dominant
negative effect to the wild-type AtSARl (MTYA1) but not
to the yeast wild type (ANY21). A similar effect was observed with NtSARl D29G. These results are summarized
in Table 2.
To examine whether these growth phenotypes were
due to the defect in the secretory pathway, the intracellular
transport of a yeast vacuolar protein, carboxypeptidase Y
(CPY), was examined by a pulse-chase experiment (Fig. 8).
In the wild-type yeast cells (ANY21), CPY undergoes sequential modification and processing, which are clearly detected by SDS-PAGE: pi (ER form) -> p2 (Golgi form) —
m (vacuolar mature form). In the ANY21/NISAR1 H74L
cells, a significant delay of transport was observed at the
step from pi to p2. Thus, the expression of this plant mutant SARI dominantly inhibits the normal function of
yeast SARI in the ER-to-Golgi transport. Such a dominant
effect was not observed when the wild-type NtSARl was expressed. Similar experiments were also attempted with
MTYNl/MS,4/?7 H74L and MTYAWAtSARl N129I cells,
but as these cells had already showed retardation of the
ER-to-Golgi transport, the effect of the expression of the
mutant SARI cDNA was not obvious (data not shown).
Discussion
In order to construct a yeast-plant shuttle system to investigate the roles of protein secretion in plant cells, we isolated the NtSARl cDNA from N. tabacum. This is a functional tobacco counterpart of SARI, because it can replace
the function of yeast SARI in the sar7-deletion mutant. We
could also show that Arabidopsis AtSARl cDNA complements the yeast sari mutant as well. Although many homologues of SARI have been found and characterized in
higher eukaryotes (d'Enfert et al. 1992, Shen et al. 1993,
Kuge et al. 1994, Davies 1994, Bar-Peled and Raikhel 1997,
Kim et al. 1997), this is the first report to show that alien
SARI genes can function in yeast cells. Interestingly, the
complementation of the yeast sari deletion mutant by
NtSARl or AtSARl is temperature sensitive. The plant5^4/?/-dependent yeast cells can grow up to 35°C but not at
37°C. This is not due to the degradation or instability of
plant Sarlp at high temperature. The intracellular pool of
plant Sarlp was quite stable even at 37°C and the purified
plant Sarlp showed appreciable GDP and GTP binding activities at low and high temperatures (M. Tada and A.
Nakano, unpublished observations). Perhaps the molecular interactions between plant Sarlp and various regulatory
components are not sound enough to sustain yeast growth
at high temperatures. In support of this, we have found
that some yeast genes that are known to suppress yeast sari
ts mutations can also suppress the ts growth of plantS/l/?7-dependent cells (M. Tada, Y. Saito and A. Nakano,
unpublished).
The predicted amino acid sequence of NtSarlp shows
quite high similarity to other members of the Sari family.
Genomic Southern blot analysis indicates that several more
homologues of NtSARl exist in BY-2 cells. Considering
the fact that multiple SARI genes have been registered
in the database for A. thaliana, Brassica campestris and
N. tabacum, as well as for Chinese hamster ovary cells, it is
almost certain that SARI constitutes a small gene family in
higher eukaryotes. Whether or not these members of the
SARI family differentiate in cellular functions would be a
quite interesting question to be addressed in the future. The
expression of NtSARl was almost ubiquitous in organs of
tobacco plant, but there was a significant difference in BY-2
cells between the exponentially growing phase and the
stationary phase. A mechanism may exist to elevate the
NtSARl expression in actively growing cells.
During the complementation tests of NtSARl and AtSARl in yeast mutant cells, we realized that the 5-noncoding region of cDNA hampers the expression of plant
cDNAs in yeast. This was especially serious in the case of
AtSARl. Unless the upstream region of the AtSARl ORF
was removed, complementation of the sari mutant was not
observed at all. It should be also mentioned that even with
the constructs that contain only ORFs, AtSARl showed
much weaker suppression activity than NtSARl towards
the seel2 ts mutation. Since AtSarlp and NtSarlp differ
only in 14 amino acid residues and 6 of them are conservative changes, this might provide an interesting clue to analyze the molecular basis of Sarlp-Secl2p interaction.
The ultimate goal of this study is to establish a plant
system, in which dominant negative mutations of plant
SARI can be conditionally expressed. For this approach to
work, it has to be guaranteed that the constructed plant mutant genes in fact act dominantly over the wild-type gene.
This is testable in yeast cells. We could demonstrate that
the expression of NtSARl D29G, NtSARl H74L, AtSARl
T51A or AtSARl N129I arrests cell growth in plant-SARldependent yeast cells (see Table 2). NtSARl H74L and AtSARl N129I were dominant lethal even in the wild-type
yeast cells. We further showed that this dominant effect in
growth was accompanied by the arrest of intracellular membrane trafficking at the step of ER-to-Golgi transport.
Thus, these mutant versions of plant SARI are quite promising tools to block secretion in plant cells. We have
already constructed plasmids containing these mutant
SARI cDNAs under regulatable plant promoters. The analysis of the effect of their expression is now under way and
will be published elsewhere.
Tobacco SARI in vesicular traffic
We are grateful to Toshiyuki Nagata, Yohsuke Takahashi
and other members of Nagata's laboratory of the University of
Tokyo for tobacco plants and the cultured BY-2 cell line, a cDNA
library from BY-2 cells, and valuable help and suggestions
throughout this work. We also thank Christophe d'Enfert of
Pasteur Institute, Takashi Ueda, Hiroshi Qadota and Yoshikazu
Ohya of the University of Tokyo, Toshihiko Oka of Osaka University, and Yumiko Saito of RIKEN for plasmids and strains, and
other members of Nakano's laboratory for comments on the manuscript. This work was supported in part by a Grant-in-Aid for Exploratory Research from the Ministry of Education, Science,
Sports and Culture of Japan and by a Special Coordination Fund
for promoting Science and Technology from the Science and Technology Agency of Japan.
References
Barlowe, C , d'Enfert, C. and Schekman, R. (1993) Purification and characterization of Sarlp, a small GTP-binding protein required for transport vesicle formation from the endoplasmic reticulum. J. Biol. Chem.
268: 873-879.
Barlowe, C. and Schekman, R. (1993) SEC12 encodes a guaninenucleotide-exchange factor essential for transport vesicle budding from
the ER. Nature 365: 347-349.
Barlowe, C , Orci, L., Yeung, T., Hosobuchi, M., Hamamoto, S.,
Salama, N., Rexach, M.F., Ravazzola, M., Amherdt, M. and Schekman, R. (1994) COPII: a membrane coat formed by Sec proteins that
drive vesicle budding from the endoplasmic reticulum. Cell 77: 895-907.
Bar-Peled, M. and Raikhel, N.V. (1997) Characterization of AISEC12 and
AtSARl. Plant Physiol. 114: 315-324.
Boeke, J.D., LaCroute, F. and Fink, G.R. (1984) A positive selection for
mutants lacking orotidine-5'-phosphate decarboxylase activity in yeast:
5-fluoro-orotic acid resistance. Mol. Gen. Genet. 197: 345-346.
Bullock, W.O., Fernandez, J.M. and Short, J.M. (1987) XLl-blue: a high
efficiency plasmid transforming recA Escherichia coli strain with beta-galactosidase selection. Biotechniques 5: 376.
Cheon, C.I., Lee, N.G., Siddique, A.B., Bal, A.K. and Verma, D.P.
(1993) Roles of plant homologs of Rablp and Rab7p in the biogenesis of
the peribacteroid membrane, a subcellular compartment formed de novo
during root nodule symbiosis. EMBO J. 12: 4125-4135.
Davies, C. (1994) Cloning and characterization of a tomato GTPase-like
gene related to yeast and Arabidopsis genes involved in vesicular transport. Plant Mol. Biol. 24: 525-531.
d'Enfert, C , Gensse, M. and Gaillardin, C. (1992) Fission yeast and a
plant have functional homologues of the Sari and Sec 12 proteins involved in ER to Golgi traffic in budding yeast. EMBO / . 11: 4205-4211.
Fabry, S., Steigerwald, R., Bernklau, C , Dietmaier, W. and Schmitt, R.
(1995) Structure-function analysis of small G proteins from Volvox and
Chlamydomonas by complementation of Saccharomyces cerevisiae
YPT/SEC mutations. Mol. Gen. Genet. 247: 265-274.
Ferro-Novick, S. and Novick, P. (1993) The role of GTP-binding proteins
in transport along the exocytic pathway. Annu. Rev. Cell Biol. 9: 575599.
Ito, H., Fukuda, Y., Murata, K. and Kimura, A. (1983) Transformation of
intact yeast cells treated with alkali cations. J. Bacteriol. 153: 163-168.
Keszenman-Pereyra, D. and Hieda, K. (1988) A colony procedure for transformation of Saccharomyces cerevisiae. Curr. Genet. 13: 21-23.
Kim, W.Y., Cheong, N.E., Je, D.Y., Kim, M.G., Lim, C O . , Bahk, J.D.,
Cho, M.J. and Lee, S.Y. (1997) The presence of a SARI gene family in
Brassica that suppresses a yeast vesicular transport mutation secl2-l.
Plant Mol. Biol. 33: 1025-1035.
Kim, W.Y., Cheong, N.E., Lee, D.C., Lee, K.O., Je, D.Y., Bahk, J.D.,
Cho, M.J. and Lee, S.Y. (1996) Isolation of an additional soybean
599
cDNA encoding Ypt/Rab-related small GTP-binding protein and its
functional comparison to Sypt using a yeast yptl-l mutant. Plant Mol.
Biol. 31: 783-792.
Kuge, O., Dascher, C , Orci, L., Rowe, T., Amherdt, M., Plutner, H.,
Ravazzola, M., Tanigawa, G., Rothman, J.E. and Balch, W.E. (1994)
Sari promotes vesicle budding from the endoplasmic reticulum but not
Golgi compartments. /. Cell. Biol. 125: 51-65.
Linsmaier, E.M. and Skoog, F. (1965) Organic growth factor requirements
of tobacco tissue cultures. Physiol Plant 18: 100-127.
Loraine, A.E., Yalovsky, S., Fabry, S. and Gruissem, W. (1996) Tomato
RablA homologs as molecular tools for studying Rab geranylgeranyl
transferase in plant cells. Plant Physiol. 110: 1337-1347.
Murray, M.G. and Thompson, W.F. (1980) Rapid isolation of high molecular weight plant DNA. Nucl. Acids. Res. 8: 4321-4325.
Nagata, T., Nemoto, Y. and Hasezawa, S. (1992) Tobacco BY-2 cell lines
as the "HeLa" cell in the cell biology of higher plants. Intl. Rev. Cytol.
132: 1-29.
Nakano, A., Brada, D. and Schekman, R. (1988) A membrane glycoprotein, Secl2p, required for protein transport from the endoplasmic
reticulum to the Golgi apparatus in yeast. J. Cell Biol. 107: 851-863.
Nakano, A. and Muramatsu, M. (1989) A novel GTP-binding protein,
Sarlp, is involved in transport from the endoplasmic reticulum to the
Golgi apparatus. J. Cell. Biol. 109: 2677-2691.
Nakano, A., Otsuka, H., Yamagishi, M., Yamamoto, E., Kimura, K.,
Nishikawa, S. and Oka, T. (1994) Mutational analysis of the Sari protein, a small GTPase which is essential for vesicular transport from the
endoplasmic reticulum. J. Biochem. 116: 243-247.
Nishikawa, S. and Nakano, A. (1991) The GTP-binding Sari protein is localized to the early compartment of the yeast secretory pathway. Biochem. Biophys. Ada 1093: 135-143.
Ohya, Y., Goebl, M., Goodman, L.E., Petersen-BJ0rn, S., Friesen, J.D.,
Tamanoi, F. and Anraku.Y. (1991) Yeast CALl is a structural and functional homologue to the DPR1 (RAM) gene involved in ras processing.
/. Biol. Chem. 266: 12356-12360.
Oka, T., Nishikawa, S. and Nakano, A. (1991) Reconstitution of GTPbinding Sari protein function in ER to Golgi transport. J. Cell. Biol.
114: 671-679.
Oka, T. and Nakano, A. (1994) Inhibition of GTP hydrolysis by Sarlp
causes accumulation of vesicles that are a functional intermediate of the
ER-to-Golgi transport in yeast. J. Cell. Biol. 124: 425-434.
Park, Y.S., Song, O., Kwak, J.M., Hong, S.W., Lee, H.H. and Nam,
H.G. (1994) Functional complementation of a yeast vesicular transport
mutation yptl-l by a Brassica napus cDNA clone encoding a small GTPbinding protein. Plant Mol. Biol. 26: 1725-1735.
Pryer, N.K., Wuestehube, L.J. and Schekman, R. (1992) Vesicle-mediated
protein sorting. Annu. Rev. Biochem. 61: 471-516.
Sambrook, J., Fritsch, E.F. and Maniatis, T. (1989) Molecular cloning: a
Laboratory Manual., 2nd ed. Cold Spring Harbor Laboratory Press,
Cold Spring Harbor, New York.
Shen, K.A., Hammond, C M . and Moore, H.-P.H. (1993) Molecular analysis of S/17?/-related cDNAs from a mouse pituitary cell line. FEBS Lett.
335: 380-385.
Sikorski, R.S. and Hieter, P. (1989) A system of shuttle vectors and yeast
host strains designed for efficient manipulation of DNA in Saccharomyces cerevisiae. Genetics 122: 19-27.
Ueda, T., Matsuda, N., Anai, T., Tsukaya, H., Uchimiya, H. and
Nakano, A. (1996) An Arabidopsis gene isolated by a novel method for
detecting genetic interaction in yeast encodes the GDP dissociation inhibitor of Ara4 GTPase. Plant Cell 8: 2079-2091.
Yamanushi, Y., Hirata, A., Oka, T. and Nakano, A. (1996) Characterization of yeast sari temperature-sensitive mutants, which are defective in
protein transport from the endoplasmic reticulum. /. Biochem. 120:
452-458.
Yoshihisa, R., Barlowe, C. and Schekman, R. (1993) Requirement for a
GTPase-activating protein in vesicle budding from the endoplasmic
reticulum. Science 259: 1466-1468.
(Received October 29, 1997; Accepted March 17, 1998)