Download RMA1, an Arabidopsis thaliana Gene Whose cDNA Suppresses the

Plant Cell Physiol. 39(5): 545-554 (1998)
JSPP © 1998
RMA1, an Arabidopsis thaliana Gene Whose cDNA Suppresses the Yeast
seel5 Mutation, Encodes a Novel Protein with a RING Finger Motif and a
Membrane Anchor
Noriyuki Matsuda1'2 and Akihiko Nakano '
' Molecular Membrane Biology Laboratory, RIKEN (The Institute of Physical and Chemical Research), Wako, Saitama, 351-0198 Japan
Institute of Molecular and Cellular Biosciences, University of Tokyo, Yayoi, Bunkyo-ku, Tokyo, 113-0032 Japan
2
To identify molecules that function in the plant secretory pathway, we screened for Arabidopsis thaliana
cDNA clones that complement the temperature-sensitive
(ts), secretion-deficient seclS mutation of yeast Saccharomyces cerevisiae. RMA1, one of the genes obtained in
this screening, suppressed not only the ts growth of sec 15
but also its secretory defect. RMA1 is not a structural homologue of SEC15 but encodes a novel 28 kDa protein
with a RING finger motif and a C-terminal membraneanchoring domain. Mutational analysis indicates that the
RING finger motif of RMA1 is important for its suppression activity. In Arabidopsis plant, RMA1 is ubiquitously
expressed. A search for homologous proteins in the database revealed that Arabidopsis, nematode, mouse and
human possess close homologues of RMA1.
Key words: Arabidopsis thaliana — Membrane protein —
Protein secretion — Saccharomyces cerevisiae.
Secretion constitutes a major mechanism for the biogenesis of cell surface in all eukaryotic organisms. A variety of newly synthesized proteins are exported from the
endoplasmic reticulum (ER) to the plasma membrane via
the Golgi apparatus by vesicular transport. Extensive genetic and biochemical studies have revealed that a great number of gene products are required as components of the
secretory machinery and that their structures and functions
are very well conserved from yeast to mammalian cells (for
reviews, see Bennett and Scheller 1993, Ferro-Novick and
Jahn 1994). However, only a little information is available
at present on the secretory mechanisms in plants.
Because plant cells cannot migrate in general, the directions of cell division and elongation are critical parameters
to determine the shape of tissues and organs in plants. The
secretory pathway is essential for the biogenesis of cell surface and cell plates and thus must be very important for
plant morphogenesis. Evidence to support this idea has
been recently provided by a genetic approach in Arabidopsis thaliana. Two A. thaliana genes, GNOM (EMB30)
and KNOLLE, whose mutations had been known to cause
aberrant pattern formation early in the embryogenesis
(Mayor et al. 1991, 1993), turned out to encode homologues of Gealp/Gea2p and syntaxin, respectively (Shevell et
al. 1994, Lukowitz et al. 1996, Busch et al. 1996). Gealp
is a yeast protein functioning as a guanine-nucleotide
exchange factor for Arflp, a small GTPase required for
vesicular traffic (Peyroche et al. 1996). Syntaxin is a synaptic membrane protein, which is essential for neurotransmitter release in neuron cells. Cognates of syntaxin, a well conserved family of proteins which are now collectively called
t-SNAREs, are essential for specific recognition between
vesicles and target membranes and thus for membrane fusion (for reviews, see Rothman 1994, Hay and Scheller
1997). Expression pattern of KNOLLE and electron micrographs of the knolle mutant suggest that Knolle is a cellplate-specific t-SNARE (Lukowitz et al. 1996). Despite
these intriguing examples, however, the roles of secretion
in plants remain largely unknown.
To obtain tools to approach many problems in the
plant secretory pathway, we decided to screen for plant
genes that complement the defects of yeast secretory mutants. Such a functional screening has been successful in
isolating plant counterparts of SEC12, SARI, PEP12 and
VAM3 from A. thaliana (d'Enfert et al. 1992, Bassham et
al. 1995, Sato et al. 1997).
Using an A. thaliana cDNA library under control of
the yeast GAL1 promoter, we isolated several clones that
complemented the seel5 mutation. SEC15 is one of the
late-acting SEC genes, which are involved in the transport
from the Golgi apparatus to the plasma membrane in
yeast. SEC15 encodes a subunit of "exocyst", a large multiprotein complex required for exocytosis (Salminen and
Novick 1989, TerBush et al. 1996). The temperature-sensitive seclS mutation is suppressed by the increase of gene
dosages of other late SEC genes, such as SEC1, SEC4,
SSO1 and SSO2 (Salminen and Novick 1989, Aalto et al.
1993). In this paper, we report the characterization of one
A. thaliana gene whose cDNA complements seel5. This
gene, named RMA1, is not homologous to SEC15 and is
thus regarded as an Arabidopsis suppressor of the yeast
Abbreviations: BCP, bromocresol-purple; BSA, bovine
serum albumin; HA, influenza hemagglutinin epitope; MC,
Wickerham's minimum medium plus 0.5% casamino acids.
The complete nucleotide sequence of RMA1 and its deduced
amino acid sequence in this paper have been submitted to DDBJ,
EMBL and GenBank under accession number AB0O8518.
545
546
Novel RING finger protein implicated in secretion
sec 15 mutation. RMA1 encodes a novel membrane protein
with a special type of zinc finger motif called "RING
finger".
Materials and Methods
Materials and culture conditions—Yeast Saccharomyces cerevisiae strains used in this study are ANS15-5B (MA Ta secl5-l ura3
Ieu2 trpl his; Nakano and Muramatsu 1989), ANY21 (MATa ura3
Ieu2 trpl his3 his4 suc2 gal2; Nakano and Muramatsu 1989), and
YPH499 (MA Ta ura3 Ieu2 his3 trpl ade2 Iys2; Sikorski and Hieter
1989). Yeast cells were grown in YPD medium [1% (w/v) Bacto
yeast extract (Difco Laboratories), 2% (w/v) polypeptone (Nihon
Seiyaku) and 2% (w/v) glucose] or in MCD [0.67% yeast nitrogen
base without amino acids (Difco), 0.5% casamino acids (Difco)
and 2% glucose] medium supplemented appropriately. YPGS and
MCGS media are YPD and MCD media containing 5% galactose
and 0.2% sucrose instead of 2% glucose, respectively. BCP/sucrose plate contained 0.003% bromocresol-purple, 0.67% yeast nitrogen base without amino acids, 0.5% casamino acids, 5% galactose, and 2% sucrose. A. thaliana (Columbia ecotype) was grown
on 0.46% Murashige-Skoog modified medium (Wako) containing
2% sucrose, 0.3% thiamine-HCl, and 0.1% vitamin stock solution (0.3% thiamine-HCl, 0.5% nicotinic acid and 0.05% pyridoxine-HCl), whose pH was adjusted to 6.3 by KOH solution.
Screening for cDNA clones that complement the secl5 mutation—A. thaliana cDNA library to be expressed in yeast was
constructed as reported previously (Ueda et al. 1996). An S. cerevisiae strain carrying the seel5-1 mutation (ANS15-5B) was transformed with this cDNA library by a lithium thiocyanate method
(Keszenman-Pereyra and Hieda 1988). The transformants (ca.
105) were cultured at 23°C for 2 d on the MCGS medium and further incubated at 37°C for 5 d. Colonies of temperature-resistant
cells were picked up and subjected to the isolation of the cDNAlibrary-derived plasmid. The plasmid dependency of the temperature-resistant phenotype was examined by retransformation of the
seclS cells. The DNA sequence of the obtained clones was determined by the dideoxy chain termination method (Sanger et al.
1977) using a DNA sequencer (Model 373A; Applied Biosystems).
Homology search was performed on the NCBI BLAST server at
both nucleotide and amino acid levels.
Subcellular fractionation—For the subcellular localization
analysis of the Rmal protein in yeast, an epitope-tagged version
of RMA1 was constructed. To insert the influenza virus hemagglutinin (HA) epitope at the N-terminus, an Nhel site was created
just after the initiation codon of RMA1 by PCR with primers:
5'-GACCACTGATGGCCGCTAGCTTAGATCAATCTTTTG-3'
and 5'-CAAAAGATTGATCTAAGCTAGCGGCCATCAGTGGTC-3'. The DNA cassette encoding three tandem repeats of the
HA epitope was excised from pYTl 1 by Nhel digestion (Takita et
al. 1995), and inserted into the Nhel site of the above construction. The expression of 3HA-RMA1 was confirmed by immunoblotting with the monoclonal anti-HA antibody 16B12 (Berkeley
Antibody).
Fractionation was performed by the method of Sato et al.
(1995). The seclS cells (ANS15-5B) harboring 3HA-RMA1 or vector alone were grown to 1 x 107 cells ml" 1 in MCGS medium. Cells
(1 x 1010) were harvested, spheroplasted, resuspended in 10 ml of
ice-chilled lysis buffer (Sato et al. 1995), and homogenized with a
Dounce homogenizer. The homogenates were centrifuged to generate low speed (13,000x g ) pellet (P13), high-speed (100,000xg)
pellet (PI00) and high-speed supernatant (SI00). These fractions
were analyzed by immunoblotting with anti-Secl2p, anti-Kex2p,
anti-Pgklp, and anti-HA antibodies.
Site-directed mutagenesis in the RING finger motif of15c-10
—The C63S point mutation of RMA1 was created by PCR with
primers: 5'-GTAATACGACTCACTATAGGGAATATTAAGC3' and 5'-GCCAGCAAAAGAGGTGACCAGAGAGAGTCAC3'. The 360-bp Kpnl-BspEll fragment of the PCR product was
used to replace the corresponding region of RMA1 to yield the
RMAlciis mutant allele. 3HA-RMAlcas
was constructed by the
same way as described above.
Southern blot analysis—Genomic DNA prepared from
A. thaliana was digested with appropriate restriction enzymes,
electrophoresed in a 0.7% agarose gel, and transferred to a nylon
membrane filter (Hybond-N + , Amersham) by the alkali method
according to the manufacture's instructions. As a probe, the
RMA1 cDNA was digested with Kpnl and Nhel to remove poly
(A) and labeled with 32P using random primers. The filter was hybridized with this probe in a solution containing 6 x SSPE (1 x
SSPE is 0.18 M NaCl, 0.01 M sodium phosphate, and 1 mM Na2
EDTA, pH 7.7), 5 x Denhardt's solution (1 x Denhardt's solution
is 0.02% Ficoll, 0.02% polyvinylpyrrolidone, and 0.02% BSA),
0.5% SDS, and 20 n% ml" 1 of salmon sperm DNA at 65°C for 16
h. The filter was washed twice with 2 x SSPE and 0.1% SDS for 5
min each at room temperature and twice for 30 min each at 65°C,
and autoradiographed. An imaging plate scanner BAS1000 (Fuji
Film) was used for visualization.
Northern blot analysis—Total RNA was isolated from Arabidopsis suspension-cultured cells (kindly provided by M. Umeda of
the University of Tokyo), roots, cotyledons, seedlings, rosettes,
stems, young siliques, and floral buds of A. thaliana by the
phenol-SDS method (Palmiter 1974). After electrophoresis of 10
H% RNA per lane in a 1.2% formaldehyde-agarose gel, RNA was
transferred to a Hybond-N + membrane. The filter was hybridized
with the same probe as above in a solution containing 50% formamide, 6 x SSPE, 5 x Denhardt's solution, 0.5% SDS, and 20
fig, ml" 1 of salmon sperm DNA at 42°C for 16 h. Thefilterwas
washed twice with 2 x SSPE and 0.1% SDS for 5 min each at
room temperature and twice for 30 min each at 65°C, and autoradiographed using BAS1000.
Results
An A. thaliana cDNA clone that complements the
secretory defect of the yeast secl5-l mutation—A cDNA
library, constructed from young seedlings of A. thaliana as
described previously (Ueda et al. 1996), was used for the
screening of genes that complement yeast sec mutants harboring a defect in the late step of the secretory pathway.
Arabidopsis cDNAs were inserted downstream of the yeast
regulatable GAL1 promoter, which is derived from the
yeast galactokinase gene and drives high expression in the
presence of galactose in the medium (Oshima 1982). Yeast
cells carrying a temperature-sensitive mutation in late-acting SEC genes (SEC1, SEC4, SEC7, SEC8, SEC10, and
SEC15) were transformed with this cDNA library. We
screened approximately 105 clones of cDNA library for
complementation of each sec mutant on galactose at restrictive temperatures. The candidate cDNA clones were recovered from the growing transformants and reintroduced
into the original mutant cells. We were able to obtain
Novel RING finger protein implicated in secretion
clones that reproducibly confer growth at the restrictive
temperature (37°C) only for the secl5-l strain (ANSISSB). Among about 30 clones obtained in this second
screen, one clone (15c-10) showed a partial but clear complementation activity reproducibly (Fig. 1A) and was subjected to further analysis. Clone 15c-10 did not complement secl-1, sec2-56, sec4-2, sec5-24, sec6-4, sec7-l, sec8-6,
sec9-4, sec 10-2, or secl4-3.
To examine whether 15c-10 complements not only the
temperature-sensitive growth defect but also the secretory
defect of the secl5 mutation, secretion of invertase (encoded by SUC2) was analyzed. The extracellular invertase activity can be detected by the change of color on a bromocresolpurple (BCP)/sucrose plate. As shown in Fig. IB, the
invertase-secreting YPH499 cells (SUC2 SEC15) changed
the color of the BCP/sucrose plate from purple to yellow
(the color change is seen as lightening in the figure), but
the suc2~ ANY21 cells (suc2 SEC15) did not. When the
ANS15-5B cells with vector alone {SUC2 seel5-1) were incubated at 37°C, the plate remained purple because invertase
was not secreted due to the seclS mutation. In contrast,
when the ANS15-5B cells (SUC2 secl5-l) harboring the
15c-10 clone were streaked, the color of the plate turned
yellow. This result indicates that 15c-10 in fact restores the
secretory activity and consequently complements the temperature-sensitive growth defect of the secl5 mutation.
15c-10 (RMA1) encodes a novel membrane protein
with a RING finger motif—The complete nucleotide sequence of 15c-10 (1,032 bp) was determined. 15c-10 shows
no sequence similarity to SEC15, meaning that it is a suppressor of the seclS mutation. The longest ORF of 15c-10
encodes a novel protein of 249 amino acid residues with a
calculated molecular mass of 28.0 kDa (Fig. 2A). This ORF
is probably full length because it is flanked by an in-frame
opal stop codon (UGA) in the 5-untranslated region at the
position of —9 to —7. The amino acid sequence deduced
from this ORF of 15c-10 unveils a special type of zinc finger
motif called "RING finger" in its N-terminal half. The typical RING finger motif is comprised of seven cysteine residues and one histidine residue which coordinate two Zn 2+
ions (for a review, see Saurin et al. 1996). The motif in 15c10 completely matches this consensus. Hydropathy analysis by the method of Kyte and Doolittle (1982) revealed that
15c-10 has a highly hydrophobic carboxyl-terminal region
of 16 amino acid residues, which is enough to span the
membrane (Fig. 2B). There is no typical signal sequence for
membrane translocation at the N-terminus. Taken together, 15c-10 was predicted to encode a membrane protein,
which is mostly facing the cytoplasm and anchored in a
membrane with the C-terminal tail, and was thus designated RMA1 for .Ring finger protein with Membrane /Inchor.
To examine the subcellular localization of the RMA1
gene product (Rmalp) in yeast, a 3HA-tagged version of
RMA1 was constructed. The HA epitope is composed of
547
15c-10
w.t.
vector
Gal
15c-10
vector
B
suc2
SEC15
SUC2
SEC15
SUC2
secl5-l
SUC2
secl5-l
15c-10
vector
Fig. 1 Complementation activity of 15c-10. A. 15c-10 complements the growth defect of the seclS mutation. Wild type cells
(YPH499), the secl5 cells (ANS15-5B) transformed with 15c-10,
and the secJ5 cells with vector alone were streaked on plates containing glucose (Glc; MCD) or galactose (Gal; MCGS) and incubated at 37°C for 4d. B. 15c-10 remedies secretory defect of the
secl5 mutation. Cells were streaked on a BCP/sucrose plate (containing galactose) and incubated at 37°C for 30 h. Used strains
were YPH499 (SUC2 SEC15), ANY21 (suc2 SEC15), and ANSISSB (SUC2 seel5) with 15c-10 or vector alone.
nine amino acid residues from human influenza hemagglutinin (Wilson et al. 1984). This 3HA-RMA1 could fully
suppress the sec15 mutation (data not shown). Immunoblotting with a monoclonal anti-HA antibody (16B12) detected a single band of 35 kDa only when 3HA-RMA1 is in-
548
Novel RING finger protein implicated in secretion
1
CTCTCGTCGAAAGCAAAACAGTCTCCTTTCTCTATCTTCTGCTCATATCAGCCATTGACACAGTTGCTTTGGGTTTCC
CTCAAACGGCGCCGATTGTCTGGATTTTGACCACTG
115/1
ATG GCC TTA GAT CAA TCT TTT GAA GAT GCT GCT TTA CTT GGA GAA CTC TAT GGA GAA GGT
Met ala leu osp gin ser phe glu asp ala ala leu leu gly glu leu tyr gly glu gly
182
20
GCA TTT TGT TTC AAG AGC AAG AAA CCT GAA CCC ATT ACA GTC TCG GTT CCT TCT GAT GAT
ala phe cys phe lys ser lys lys pro glu pro ile thr val ser val pro ser asp asp
242
40
ACT GAT GAT TCG AAT TTT GAC TGC AAT ATT TGC TTA GAC TCG GTG CAA GAA CCT GTT GTG
thr asp asp ser asn phe asp SE asn ile S B leu asp ser val gin glu pro val val
302
60
ACT CTC TGT GGT CAC CTC TTT TGC TGG CCT TGT ATT CAC AAA TGG CTT GAT GTA CAG AGC
thr leu 3 3 3 gly BE leu phe 9 9 § trp pro 9 H ile his lys trp leu asp val gin ser
362
80
TTC TCA ACA AGT GAT GAA TAC CAA AGA CAT AGA CAG TGT CCT GTT TGT AAA TCT AAA GTT
phe ser thr ser asp glu tyr gin arg his arg gin 9E pro val 9 X 3 lys ser lys val
422
100
TCT CAT TCT ACT TTG GTT CCT TTG TAT GGT AGA GGC CGT TGT ACT ACT CAG GAG GAA GGT
ser his ser thr leu val pro leu tyr gly arg gly arg cys thr thr gin glu glu gly
482
120
AAA AAC AGT GTG CCT AAA AGA CCC GTA GGA CCG GTT TAT CGG CTT GAA ATG CCG AAT TCA
lys asn ser val pro lys arg pro val gly pro val tyr arg leu glu met pro asn ser
542
140
CCT TAT GCA AGT ACT GAT CTG CGG TTA TCA CAA CGG GTT CAT TTC AAT AGC CCA CAG GAA
pro tyr ala ser thr asp leu arg leu ser gin arg val his phe asn ser pro gin glu
602
160
GGT TAC TAC CCT GTC TCA GGG GTG ATG AGC TCG AAC AGT TTA TCA TAC TCT GCT GTT TTG
gly tyr tyr pro val ser gly val met ser ser asn ser leu ser tyr ser ala val leu
662
180
GAT CCG GTG ATG GTG ATG GTT GGA GAA ATG GTA GCT ACG AGG TTG TTT GGA ACA CGA GTG
asp pro val met val met val gly glu met val ala thr arg leu phe gly thr arg val
722
200
ATG GAT AGA TTT GCG TAT CCG GAC ACT TAC AAT CTC GCA GGG ACT AGC GGG CCG AGG ATG
met asp arg phe ala tyr pro asp thr tyr asn leu ala gly thr ser gly pro arg met
782
220
AGA AGG CGG ATA ATG CAG GCA GAT AAA TCG CTG GGA AGA ATC TTC TTC TTC TTT ATG TGT
arg arg arg ile met gin ala asp lys ser leu gly arg ile phe phe phe phe met cys
842
240
TGT GTT GTT CTG TGT CTT CTC TTG TTT TAG
cys val val leu cys leu leu leu phe *
861
249
GTTTTCATAGCTAGCTTGGTTCTGCTACTGTTCAGTTTCTTCAGGTGTAAGGAAAACATAGTCAAAGAAATGTACATTTG
TGTTGGAAACAAATCAAAGTTGCTTAATGTTGAGGGAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA
AAAAAAAA 1032
B
249
amino acid residues
Fig. 2 A. Complete nucleotide and predicted amino acid sequences of 15c-10 (RMA1). Black boxes indicate the residues comprising
the RING finger motif and underline shows the membrane-anchoring domain. B. Hydropathy profile of the predicted 15c-10 protein
(Rmalp). Bar represents the membrane anchor.
Novel RING finger protein implicated in secretion
duced by galactose. The size of this band is consistent with
the predicted molecular mass of 3HA-tagged Rmalp (32
kDa). The seel5 cells harboring 3HA-RMA1 were spheroplasted, homogenized, and subjected to differential centrifugation to yield low-speed (13,000 xg) pellet (P13),
high-speed (100,000 xg) pellet (P100), and high-speed
(100,000 xg) supernatant (S100) fractions. Generally, the
membranes of ER/nuclear envelope, vacuoles, mitochondria and the plasma membrane are primarily recovered in
the PI3 fraction, whereas the Golgi membrane and transport vesicles are mainly collected in the PI00 fraction (Marcusson et al. 1994, Sato et al. 1995, Rieder and Emr 1997).
These fractions were analyzed by immunoblotting with several antibodies. Secl2p (Nakano et al. 1988) and Kex2p
(Fuller et al. 1989) were used as membrane markers of the
ER and the late compartment of the Golgi apparatus, respectively. Pgklp, 3-phosphoglycerate kinase (Hitzeman et
al. 1982), was used as a cytosolic marker. As shown in
Fig. 3, Secl2p and Kex2p were mostly fractionated in P13
and P100, respectively. Pgklp was recovered predominantly in S-100 but also in other fractions. The anti-HA antibody (16B12) detected 3HA-Rmalp in P13 and P100
m
a.
oo
a.
|
CO
Vector
anti-HA
3H A-RMA1
fractions but not in SI00 at all. By indirect immunofluorescence microscopy, the anti-HA antibody stained punctate
structures near nuclei, which did not look like the typical
patterns of ER, Golgi or secretory vesicles (data not
shown). These observations indicate that the product of
RMAl is in fact a membrane protein and is localized to
some intracellular membrane structures in yeast cells.
RMAl homologues are found in plant and animal
kingdoms—Homologues of RMAl were searched in the
database using the NCBI BLAST server. We first realized
that Rmalp shows limited but significant similarity to some
yeast proteins involved in protein transport, Pep5p, PexlOp, and Vpsl8p (Fig. 4A). All of them contain the RING
Rmal
A. thaliana
H. sapiens
M. musculus
C. elegans
Rmal
A thaliana
H. sapiens
M. musculus
C elegans
anti-HA
Rmal
A. thaliana
H. sapiens
M. musculus
C. elegans
Sec12p
Rmal
A. thaliana
H. sapiens
M. musculus
C. elegans
Kex2p
Rmal
A. thaliana
H. sapiens
M. musculus
C. elegans
Pgklp
Rmal
A. thaliana
H. sapiens
M. musculus
C. elegans
Fig. 3 Subcellular fractionation of 3HA-Rmalp. ThesecJ5 cells
with 3HA-RMA1 or vector alone were spheroplasted, homogenized, and subjected to differential centrifugation to separate P13,
P100, and S100 fractions. The obtained fractions equivalent to
2x 107 cells were analyzed by immunoblotting with anti-Sec 12p
(ER marker), anti-Kex2p (Golgi marker), anti-Pgklp (cytosol
marker), and anti-HA antibodies.
549
Fig. 4 A. Amino acid sequence comparison of Rmalp, Pep5p,
PexlOp and Vpsl8p. White boxes represent the similar amino acid
residues and black boxes indicate conserved amino acid residues.
B. Sequence comparison between RMAl and its homologues
(A. thaliana U81598, H. sapiens U89336, M. musculus AF03O0O1,
and C. elegans Z46787). Black boxes indicate conserved amino
acid residues.
S5G
Novel RING finger protein implicated in secretion
finger motif and the sequences conserved are confined in
this region. Pep5p and Vpsl8p are involved in vesicular
transport from the Golgi apparatus to the vacuole in yeast
S.cerevisiae (Woolford et al. 1990, Robinson et al. 1991,
Preston et al. 1991). PexlOp is essential for translocation of
peroxisomal proteins in yeast Pichia pastoris (Kalish et al.
1995, Distel et al. 1996). More interestingly, the homology
search in the database disclosed that A. thaliana, Homo sapiens, Mus musculus, and Caenorhabditis elegans possess
CNICLDSVQEPVVTLCGHLFCWPCIHKWLDVQSFSTSDEYQRHRQCPVC
B
RMA1
close homologues of RMA1 (Fig. 4B). Amino acid sequence identities are 40% for the A. thaliana homologue
(GenBank accession number U81598), 26% for the H. sapiens homologue (accession number U89336), 26% for the
M. musculus homologue (accession number AF030001),
and 28% for the C. elegans homologue (accession number
Z46787). The homology of these proteins extends to the entire sequences. Furthermore, they all have a typical RING
finger motif and a C-terminal membrane-anchoring domain. The Arabidopsis homologue (U81598) has been submitted to the database as a gene preferentially expressed
during seed development. Other three proteins (U89336,
AF030001, and Z46787) have been identified by genome sequencing projects. Such a close homologue of RMAJ was,
however, not present in the recently-completed genome sequence of yeast S. cerevisiae.
RING finger motif of RMA1 is essential for its suppression activity—The RING finger motifs of Vps8p and
Vpsl8p have been reported to be essential for their functions (Robinson et al. 1991, Horazdovsky et al. 1996). To
test whether it is also true for Rmalp, we changed the conserved third cysteine of the RING finger motif (C63) to
serine by site-directed mutagenesiss (Fig. 5A). Serine at this
position has been shown to reduce zinc binding activity in
vitro (Kalish et al. 1995). The constructed mutant allele,
C63S
E B H X
Vector
RMA1
i
21kb
12kb
8
S
S
>
a: a;
6kb
3kb
1.5kb
Fig. 5 Mutational analysis of the RING finger domain of
RMA1. A. The mutation (C63S) introduced in the RING finger
motif. The eight amino acid residues which are thought to coordinate zinc ions are highlighted. B. The secJ5 cells harboring vector,
wild-type RMA1, or mutant RMAI(C63S) were streaked on an
MCGS plate and incubated at 37°C for 3 d. C. The secl5 cells carrying vector, 3HA-RMA1, and 3HA-RMAJ(C63S) were incubated at 37°C for 2 h and subjected to immunoblotting with anti-HA
antibody.
*
0.5kbFig. 6 Southern blot analysis of RMA1. Genomic DNA was prepared from A. thaliana, digested with EcoRl (E), BamHl (B), Hindlll (H), and Xhol (X), and subjected to Southern hybridization.
The full-length RMA1 cDNA fragment without the poly (A)-tail
was used as a probe.
Novel RINGfingerprotein implicated in secretion
RMA1C63S, was introduced into the seel5 mutant cells and
tested for the suppression activity. As shown in Fig. 5B, the
wild type RMA1 clearly suppressed the secl5 mutation, but
the mutant RMAlC6iS did not. No toxic effect was observed
when the transformant with RMA1C61S was incubated
at 23°C (data not shown). To rule out the possibility that
the inability of Rmalp C63S to suppress seel5 was due to its
low expression, we constructed a 3HA-tagged version of
RMA1C63S and introduced it into the seclS mutant cells. Immunoblotting showed that the 3HA-tagged Rmalp C63S was
almost equally expressed as the 3HA-tagged wild-type
Rmalp (Fig. 5C), but again unable to suppress secl5 (not
shown). From these results, we concluded that the RING
finger motif of RMA1 was essential for its suppression activity toward seclS.
Southern and Northern blot analyses of RMA1—
Genomic DNA was prepared from A. thaliana, digested
with EcoRl, BamHl, Hindlll or Xho\, separated in an
agarose gel and hybridized with the full-length cDNA of
RMA1. This probe contains one restriction site each for
EcoRl and BamHl but none for Hindlll and Xhol. As
shown in Fig. 6, single bands were observed in the lanes of
Hindlll and Xhol, and two bands were seen in the lanes of
EcoRl and BamHl. This result indicates that RMA1 exists
at a single locus in the Arabidopsis genome.
S51
The expression of RMA1 in various organs of Arabidopsis plant was examined by Northern blot analysis. Total
RNA was prepared from suspension-cultured cells, roots,
cotyledons, seedlings, rosettes, stems, young siliques, and
floral buds of A. thaliana, separated in an agarose gel and
was hybridized with the RMA1 cDNA probe (Fig. 7). A
single band of 1.4 kb, which corresponds to the RMA1
transcript, was observed in every lane. The expression level
was low but detectable in the all examined organs of Arabidopsis plant and higher in suspension-cultured cells.
Discussion
For the purpose of obtaining molecular tools to exploit the roles of protein secretion in plants, we screened an
A. thaliana cDNA library for clones that complement yeast
sec mutants. RMA1, the cDNA clone we isolated as the one
complementing seel5, restores not only the temperaturesensitive growth but also the secretion defect of the seclS
mutant cells.
The DNA sequence of RMA1 reveals that it encodes a
novel protein of 249 amino acid residues containing a
RING finger motif and a C-terminal membrane anchor. It
is not similar to the yeast SEC15 gene at all and has no
homologous counterpart in the yeast genome. Thus, the
expression of RMA1 in yeast appears to have suppressed secl5 fortuitously. However, we would propose that
RMA1 plays some important role in A. thaliana perhaps in
the secretory pathway for the reasons we will discuss
hereafter.
Connection between RMA1 and SEC15—Yeast SEC15
RMA1
is a gene encoding a subunit of "exocyst," a large cytosolic
protein complex required for exocytosis. This complex
- 3.4 kb
contains Sec3p, Sec5p, Sec6p, Sec8p, SeclOp and Exo70p
in addition to Secl5p (TerBush et al. 1996). Strong genetic
interactions have been reported among these genes. For
1.8 kb
example, the combination of sec3, sec5, sec8 or seclO
with sec 15 leads to synthetic lethality. Such genetic relationships with SEC15 can be also seen with SEC2, SEC4, SSO1
and SSO2. SEC4 encodes a small GTPase of the Rab/Ypt
family, a key molecule of the recognition between secretory
vesicles and the plasma membrane (Salminen and Novick
1987). The elevation of the gene dosage of SEC4 can suppress
the ts defect of seclS and the sec4 and seclS double
18S
mutant is lethal. The product of SEC2 is a protein catalyzing guanine nucleotide exchange of Sec4p (Walch-Solimena
et al. 1997). sec2 is also synthetically lethal with seclS.
SSO1 and SSO2 code for t-SNAREs on the plasma membrane, whose overproduction suppresses seel5 (Aalto et al.
1993). The intimate genetic interactions among these molecules
indicate that they cooperate in the correct targeting
Fig. 7 Expression of RMA1. Total RNA (10 fi%) extracted from
various tissues of A. thaliana was subjected to Northern blot analy- and fusion of secretory vesicles with the plasma membrane
sis. The full-length RMA1 cDNA fragment without the poly (A)- and that their functions can be modulated by complex
tail was used as a probe. The blot of 18S Ribosomal RNA is also mutual interactions.
shown as a positive control.
552
Novel RING finger protein implicated in secretion
The RING finger motif was first discovered in a transcription factor and has been claimed to represent a class of
common motifs found in many DNA binding proteins
(Freemont et al. 1991). However, it is unlikely that Rmalp
itself functions as a transcription factor for the following
two reasons. First, Rmalp does not have any typical DNA
binding motif as the RING-finger-containing transcription
factors do. Second, Rmalp has a stretch of hydrophobic
amino acid residues at the C-terminus, which is predicted
to act as a membrane anchor. Rmalp is in fact associated
with membranes when expressed in yeast cells. Although
there is an example that a membrane protein can play a role
as a transcription factor after proteolytic processing and
translocation into the nucleus (Wang et al. 1994), we could
not detect any soluble derivatives of Rmalp in yeast cells.
Another important feature of the RING finger motif is
that it is found in the proteins that interact with small
GTPases. For example, Vps8p, a yeast protein with the
RING finger motif involved in vacuolar protein sorting,
has been shown to function together with a small GTPase,
Vps21p (Horazdovsky et al. 1996). Ardlp, a mammalian
protein containing the RING-finger and Arf (small GTPase)-like domains, has been reported to display an interaction between the two domains within the molecule (Vitale
et al. 1997). If more general zinc-finger motifs are included,
there are many examples of the connection to small GTPases, such as Raf-Ras and Rabphilin-Rab3 interactions
(Hu et al. 1995, Luo et al. 1997, Stahl et al. 1996).
Furthermore, a number of RING-finger proteins are
known to be important for intracellular protein transport.
They include Vps8p, Vpsl8p, and Pep5p involved in vacuolar protein sorting and Pex2p, PexlOp and Pexl2p required
for peroxisomal protein import. Interestingly, all of these
molecules are membrane proteins (Horazdovsky et al.
1996, Erdmann et al. 1997, Rieder and Emr 1997). Thus,
Rmalp may well have a function in protein trafficking in
A. thaliana.
Even if RMA1 plays a role in secretion in A. thaliana,
how can it suppress the defect of sec!5 in yeast in which no
true counterpart of RMA1 exists? There are some precedent reports of such genetic interaction between seeminglyunrelated genes of different species. The most relevant case
may be that the expression of mammalian synaptotagmin
II partially rescues the growth defect of secl5 (Darner
and Creutz 1996). Synaptotagmin is a membrane protein
which is essential for Ca 2+ -regulated synaptic vesicle fusion
(Siidhof and Rizo 1996). It has no homologue in yeast.
Besides the fact that both synaptotagmin and Secl5p function in exocytosis, the physiological significance of the suppression of seclS by synaptotagmin is supported by the
observation that synaptotagmin expressed in yeast cells appears to be localized on secretory vesicles. They may physically encounter in yeast cells. Another intriguing example is
that the expression of human GPS1 and GPS2 genes sup-
presses the growth defect of the yeast gpal mutant (Spain
et al. 1996). The GPA1 gene encodes the a subunit of a
yeast trimeric G protein (Miyajima et al. 1987), which regulates the downstream mitogen-activated protein kinase
(MAPK)-cascade for the pheromone response (Herskowitz
1995). The yeast genome does not have any homologues of
GPS1 or GPS2. However, GPS1 and GPS2 were found to
suppress a MAPK-mediated signal when overexpressed in
mammalian cells, suggesting that they could modulate a
signal transduction pathway which may be somehow conserved between mammalian and yeast systems. In the case
of RMA1, it is also possible that RMA1 suppresses seel5
via some component(s) conserved between yeast and plant.
Considering the possible relationship between RINGfinger proteins and small GTPases, a candidate as the
target of Rmalp in yeast is the Sec4 protein. As mentioned
above, the elevation of the gene dosage of SEC4 strongly
suppresses the secl5 mutation. The expression of Rmalp in
yeast cells may increase or activate Sec4p and thus suppress
seclS. We examined the subcellular localization of Rmalp
in yeast, but could not observe its clear localization on the
secretory vesicles, where Sec4p functions. The mechanism
of suppression by Rmalp may be different from the case of
synaptotagmin.
A. thaliana has many members of the Rab/Ypt family
of GTPases. Among them, the gene which is most similar
to SEC4 is ARA3 (Anai et al. 1991). In A. thaliana, Rmalp
could interact with Ara3p and regulate its function. In our
preliminary attempt using the yeast two-hybrid method,
however, we could not show direct physical interaction between them. Further studies will be necessary to elucidate
which molecule is interacting with Rmalp in A. thaliana.
Family of RMA1—Regarding the role of the RING
finger motif, we have demonstrated that it is essential for
Rmalp to suppress seclS. In plants, five genes including
RMA1 have been so far identified to encode RING-finger
containing proteins. Other four are COP1, PZF, ATL2,
and A-RZF. Coplp, which regulates the photomorphogenesis of A. thaliana (Deng et al. 1992), and Pzfp, a putative
transcription factor of Lotus japonicus (Schauser et al.
1995), are soluble proteins. /17X2 has been isolated from
A. thaliana as the gene whose overexpression is toxic to
yeast. It is interesting that Atl2p has a putative transmembrane domain near the N-terminus, but its in vivo function
is unknown (Martinez-Garcia et al. 1996). A-RZFis also an
A. thaliana gene, whose expression seems to increase during seed development (Zou and Taylor 1997). The product
of A-RZF is homologous to Rmalp and harbors a membrane anchor at the C-terminus.
RMA1 has close homologues in various species in
higher eukaryotes. Four highly-homologous genes were
found in the database at the time of preparation of this
manuscript. They are from A. thaliana, C. elegans, mouse
and human. All of them show remarkable similarity to
Novel RINGfingerprotein implicated in secretion
RMAl for the whole sequence, implying that this family of
proteins fulfill very important cellular functions that have
been conserved during evolution. All the gene products contain not only the RING finger motif but also the C-terminal
membrane-anchoring sequence. Their exact functions, however, are unknown at the moment. The animal homologues
were identified by genome projects, whereas the Arabidopsis homologue was registered to the database as a gene
preferentially expressed during seed development. While
this manuscript was in preparation, the cloning and characterization of this Arabidopsis homologue was published
with the gene name of A-RZF (Zou and Taylor 1997). The
authors claim the possibility that its product may play a
role in gene expression in the nucleus. However, no direct
evidence was provided, and we think it unlikely for the
same reasons we mentioned above for Rmalp. If A-RZF
performs a similar function to RMA1, its possible role in
the secretory processes during seed development will be an
interesting question. In fact, seed development is known to
be accompanied by active protein transport because many
storage proteins are synthesized and transported to the
vacuole during seed maturation as carbon and nitrogen
sources for later germination (for a review, see Okita and
Rogers 1996).
Role o/RMAl—In order to elucidate the precise roles
of this family of proteins, further studies are obviously required. For example, the expression of RMA1 in A. thaliana seems to be ubiquitous and high in suspension-cultured
cells as shown by the Northern blotting experiment. This
result is consistent with the presumed function of Rmalp
because suspension-cultured cells are active in secretion to
ensure rapid cell-division and elongation. However, more
detailed analysis such as in situ hybridization remains to be
done to discuss its differential expression at cellular levels.
To understand the in vivo function of RMAl, and to examine its role in protein secretion, a variety of approaches
should be taken. The expression of the antisense RNA
would be a useful strategy. It is interesting that the overexpression of a truncated form of Coplp, which contains the
RING finger motif but lacks the C-terminal region, gives a
dominant negative effect to A.thaliana (McNellis et al.
1996), probably because it competes for normal target molecules with the wild-type Coplp. The overexpression of similar truncated forms of RMAl in A. thaliana may be a quite
powerful approach as well and is now underway.
553
We are grateful to Masahiko Sato and Yoh Wada for their
help and advice during construction of the Arabidopsis cDNA
library and to Hirofumi Uchimiya and Takashi Ueda of the University of Tokyo and the members of Nakano's laboratory
in RIKEN for valuable discussions. We also thank Masaaki
Umeda of the University of Tokyo for the suspension culture of
A. thaliana and Yoshikazu Ohya of the University of Tokyo and
Minami Matsui of RIKEN for plasmids. This work was supported
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 Promotion of Science and Technology
from the Science and Technology Agency of Japan.
References
Aalto, M.K., Ronne, H. and Keranen, S. (1993) Yeast syntaxins Ssolp and
Sso2p belong to a family of related membrane proteins that function in
vesicular transport. EMBO J. 12: 4095-4104.
Anai, T., Hasegawa, K., Watanabe, Y., Uchimiya, H., Ishizaki, R. and
Matsui, M. (1991) Isolation and analysis of cDNAs encoding small GTPbinding proteins of Arabidopsis thaliana. Gene 108: 259-264.
Bassham, D.C., Gal, S., Conceicao, A.D.S. and Raikhel, N.V. (1995) An
Arabidopsis syntaxin homologue isolated by functional complementation of a yeast pepl2 mutant. Proc. Nail. Acad. Sci. USA 92: 7262-7266.
Bennett, M.K. and Scheller, R.H. (1993) The molecular machinery for
secretion is conserved from yeast to neurons. Proc. Natl. Acad. Sci.
USA 90: 2559-2563.
Busch, M., Mayer, U. and Jiirgens, G. (1996) Molecular analysis of the
Arabidopsis pattern formation gene GNOM: gene structure and intragenic complementation. Mol. Gen. Genet. 250: 681-691.
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 J. 11: 4205-4211.
Darner, C.K. and Creutz, C.E. (1996) Synaptotagmin II expression partially rescues the growth defect of the yeast seel5 secretory mutant. Biol.
CellSS: 55-63.
Deng, X-W., Matsui, M., Wei, N., Wagner, D., Chu, A.M., Feldmann,
K.A. and Quail, P.H. (1992) COPJ, an Arabidopsis regulatory gene, encodes a protein with both a zinc-binding motif and a G0 homologous domain. CW/71: 791-801.
Distel, B. et al. (1996) A unified nomenclature for peroxisome biogenesis
factors. J. Cell Biol. 135: 1-3.
Erdmann, R., Veenhuis, M. and Kunau, W-H. (1997) Peroxisomes: organelles at the crossroads. Trends Cell Biol. 7: 400-407.
Ferro-Novick, S. and Jahn, R. (1994) Vesicle fusion from yeast to man.
Nature 370: 191-193.
Freemont, P.S., Hanson, I.M. and Trowsdale, J. (1991) A novel cysteinerich sequence motif. Cell 64: 483-484.
Fuller, R.S., Brake, A. and Thorner, J. (1989) Yeast prohormone processing enzyme (KEX2 gene product) is a Ca2+-dependent serine protease.
Proc. Natl. Acad. Sci. USA 86: 1434-1438.
Hay, J.C. and Scheller, R.H. (1997) SNAREs and NSF in targeted membrane fusion. Curr. Opin. Cell Biol. 9: 505-512.
Herskowitz, I. (1995) MAP kinase pathways in yeast: for mating and
more. Cell 80: 187-197.
Hitzeman, R.A., Hagie, F.E., Hayflick, J.S., Chen, C.Y., Seeburg, P.H.
and Derynck, R. (1982) The primary structure of the Saccharomyces
cerevisiae gene for 3-phosphoglycerate kinase. Nucl. Acids Res. 10:
7791-7808.
Horazdovsky, B.F., Cowles, C.R., Mustol, P., Holmes, M. and Emr, S.D.
(1996) A novel RING finger protein, Vps8p, functionally interacts with
the small GTPase, Vps21p, to facilitate soluble vacuolar protein localization. J. Biol. Chem. 11\: 33607-33615.
Hu, C-D., Kariya, K., Tamada, M., Akasaka, K., Shirouzu, M., Yokoyama, S. and Kataoka, T. (1995) Cysteine-rich region of Raf-1 interacts
with activator domain of post-translationally modified Ha-Ras. /. Biol.
Chem. 270: 30274-30277.
Kalish, J.E., Theda, C , Morrell, J . C , Berg, J.M. and Gould, S.J. (1995)
554
Novel RING finger protein implicated in secretion
Formation of the peroxisome lumen is abolished by loss of Pichia
pastoris Pas7p, a zinc-binding integral membrane protein of the peroxisome. Mol. Cell. Biol. 15: 6406-6419.
Keszenman-pereyra, D. and Hieda, K. (1988) A colony procedure for transformation of Saccharamyces cerevisiae. Curr. Genet. 13: 21-23.
Kyte, J. and Doolittle, R.F. (1982) A simple method for displaying the hydropathic character of a protein. / . Mol. Biol. 157: 105-132.
Lukowitz, W., Mayer, U. and Jiirgens, G. (1996) Cytokinesis in the Arabidopsis embryo involves the syntaxin-related KNOLLE gene product.
CW/84: 61-71.
Luo, Z., Diaz, B., Marshall, M. and Avruch, J. (1997) An intact Raf zinc
finger is required for optimal binding to processed Ras and for Ras-dependent Raf activation in situ. Mol. Cell. Biol. 17: 46-53.
Marcusson, E.G., Horazdovsky, B.F., Cereghino, J.L., Gharakhanian, E.
and Emr, S.D. (1994) The sorting receptor for yeast vacuolar carboxypeptidase Y is encoded by the VPSIO gene. Cell 77: 579-586.
Martinez-Garcia, M., Garciduenas-Pina, C. and Guzman, P. (1996) Gene
isolation in Arabidopsis thaliana by conditional overexpression of
cDNAs toxic to Saccharomyces cerevisiae: identification of a novel early
response zinc-finger gene. Mol. Gen. Genet. 252: 587-596.
Mayor, U., Biittener, G. and Jurgens, G. (1993) Apical-basal pattern formation in the Arabidopsis embryo: studies on the roles of the gnom
gene. Development 117: 149-162.
Mayor, U., Torres-Ruiz, R.A., Berleth, T., Misera, S. and Jurgens, G.
(1991) Mutations affecting body organization in the Arabidopsis embryo. Nature 353: 402-407.
McNellis, T.W., Torii, K.U. and Deng, X-W. (1996) Expression of an Nterminal fragment of COPl confers a dominant-negative effect on lightregulated seedling development in Arabidopsis. Plant CellS: 1491-1503.
Miyajima, I., Nakafuku, M., Nakayama, N., Brenner, C , Miyajima, A.,
Kaibuchi, K., Arai, K., Kaziro, Y. and Matsumoto, K. (1987) GPA1, a
haploid-specific essential gene, encodes a yeast homolog of mammalian
G protein which may be involved in mating factor signal transduction.
Cell 50: 1011-1019.
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. / . 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.
Okita, T.W. and Rogers, J.C. (1996) Compartmentation of proteins in the
endomembrane system of plant cells. Annu. Rev. Plant Physiol. Plant
Mol. Biol. 47: 327-350.
Oshima, Y. (1982) Regulatory circuits for gene expression: the metabolism
of galactose and phosphate. In The Molecular Biology of the Yeast Saccharomyces: Metabolism and Gene Expression. Edited by Strathern,
J.N., Jones, E.W. and Broach, J.R. pp. 159-180. Cold Spring Harbor
Laboratory Press, N.Y.
Palmiter, R.D. (1974) Magnesium precipitation of ribonucleoprotein complexes: expedient techniques for the isolation of undergraded polysomes
and messenger ribonucleic acid. Biochemistry 13: 3606-3615.
Peyroche, A,, Paris, S. and Jackson, C.L. (1996) Nucleotide exchange on
ARF mediated by yeast Geal protein. Nature 384: 479-481.
Preston, R.A., Manolson, M.F., Becherer, K., Weidenhammer, E.,
Kirkpatrick, D., Wright, R. and Jones, E.W. (1991) Isolation and characterization of PEP3, a gene required for vacuolar biogenesis in Saccharomyces cerevisiae. Mol. Cell. Biol. 11: 5801-5812.
Rieder, S.E. and Emr, S.D. (1997) A novel RING finger protein complex
essential for a late step in protein transport to the yeast vacuole. Mol.
Biol. Cell 8: 2307-2327.
Robinson, J.S., Graham, T.R. and Emr, S.D. (1991) A putative zinc finger
protein, Saccharomyces cerevisiae Vpsl8p, affects late Golgi functions required for vacuolar protein sorting and efficient a-factor prohormone
maturation. Mol. Cell. Biol. 12: 5813-5824.
Rothman, J.E. (1994) Mechanisms of intracellular protein transport.
Nature 372: 55-63.
Salminen, A. and Novick, P.J. (1987) A ras-like protein is required for a
post-Golgi event in yeast secretion. Cell 49: 527-538.
Salminen, A. and Novick, P.J. (1989) The Secl5 protein responds to the
function of the GTP binding protein, Sec4, to control vesicular traffic in
yeast. J. Cell Biol. 109: 1023-1036.
Sanger, F., Nicklen, S. and Coulson, A.R. (1977) DNA sequencing with
chain-terminating inhibitors. Proc. Natl. Acad. Sci. USA 74: 5463-5467.
Sato, K., Nishikawa, S. and Nakano, A. (1995) Membrane protein
retrieval from the Golgi apparatus to the endoplasmic reticulum (ER):
characterization of the RER1 gene product as a component involved in
ER localization of Secl2p. Mol. Biol. Cell 6: 1459-1477.
Sato, M.H., Nakamura, N., Ohsumi, Y., Kouchi, H., Kondo, M., HaraNishimura, I., Nishimura, M. and Wada, Y. (1997) The AIVAM3 encodes a syntaxin-related molecule implicated in the vacuolar assembly in
Arabidopsis thaliana. J. Biol. Chem. 272: 24530-24535.
Saurin, A.J., Borden, K.L.B., Boddy, M.N. and Freemont, P.S. (1996)
Does this have a familiar RING? Trends Biochem. Sci. 21: 208-214.
Schauser, L., Christensen, L., Borg, S. and Poulsen, C. (1995) PZF, a
cDNA isolated from Lotus japonicus and soybean root nodule libraries,
encodes a new plant member of the RING-finger family of zinc-binding
proteins. Plant Physiol. 107: 1457-1458.
Shevell, D.E., Leu, W-M., Gillmor, C.S., Xia, G., Feldman, K.A. and
Chua, N-H. (1994) EMB30 is essential for normal cell division, cell expansion, and cell adhesion in Arabidopsis and encodes a protein that has
similarity to Sec7. Cell 77: 1051-1062.
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.
Spain, B.H., Bowdish, K.S., Pacal, A.R., Staub, S.F., Koo, D., Chang,
C-Y.R., Xie, W. and Colicelli, J. (1996) Two human cDNAs, including a
homolog of Arabidopsis FUS6 (COPlI), suppress G-protein- and Mitogen-Activated Protein Kinase-mediated signal transduction in yeast and
mammalian cells. Mol. Cell. Biol. 16: 6698-6706.
Stahl, B., Chou, J.H., Li, C , Sudhof, T.C. and Jahn, R. (1996) Rab3
reversibly recruits rabphilin to synaptic vesicles by a mechanism analogous to raf recruitment by ras. EMBO J. 15: 1799-1809.
Surdhof, T.C. and Rizo, J. (1996) Synaptotagmins: C2-domain proteins
that regulate membrane traffic. Neuron 17: 379-388.
Takita, Y., Ohya, Y. and Anraku, Y. (1995) The CLS2 gene encodes a protein with multiple membrane-spanning domains that is important Ca2+
tolerance in yeast. Mol. Gen. Genet. 246: 269-281.
TerBush, D.R., Maurice, T., Roth, D. and Novick, P. (1996) The Exocyst
is a multiprotein complex required for exocytosis in Saccharomyces cerevisiae. EMBO J. 15: 6483-6494.
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.
Vitale, N., Moss, J. and Vaughan, M. (1997) Interaction of the GTP-binding and GTPase-activating domains of ARD1 involves the effector
region of the ADP-ribosylation factor domain. / . Biol. Chem. 272:
3897-3904.
Walch-Solimena, C , Collins, R.N. and Novick, P.J. (1997) Sec2p mediates nucleotide exchange on Sec4p and is involved in polarized delivery
of post-Golgi vesicles. / . Cell Biol. 137: 1495-1509.
Wang, X., Sato, R., Brown, M.S., Hua, X. and Goldstein, J.L. (1994)
SREBP-1, a membrane-bound transcription factor released by sterol-regulated proteolysis. Cell 77: 53-62.
Wilson, I.A., Niman, H.L., Houghton, R.A., Cherenson, A.R., Connolly, M.L. and Lerner, R.A. (1984) The structure of an antigenic determinant in a protein. Cell 37: 767-778.
Woolford, C.A., Dixson, C.K., Manolson, M.F., Wright, R. and Jones,
E.W. (1990) Isolation and characterization of PEP5, a gene essential for
vacuolar biogenesis in Saccharomyces cerevisiae. Genetics 125: 739-752.
Zou, J. and Taylor, D.C. (1997) Cloning and molecular characterization of
an Arabidopsis thaliana RING zinc finger gene expressed preferentially
during seed development. Gene 196: 291-295.
(Received January 5, 1998; Accepted March 9, 1998)