Curr Genet (1995) 27:123-130

9 Springer-Verlag 1995

Brigitte Pertuiset. Jean-Marie Beckerich 9Claude Gaillardin

Molecular cloning of Rab-related genes in the yeast Yarrowia lipolytica. Analysis of RYL1, an essential gene encoding a SEC4 homologue

Received: 5 April 1994/17 July 1994

Abstract Small GTP-binding proteins of the Rab family are involved in the vesicular traffic inside eukaryotic cells. A gene library from the yeast Yarrowia lipolytica was screened with an oligonucleotide deduced from a highly conserved sequence in the Rab family. Four different genes were isolated. One of them, RYL1, was shown to be essential for cell viability. R Y L l p displayed a high similarity with and tight phylogenetic relationships to SEC4p. When placed under the control of the GALIO promoter, RYL1 was able to specifically relieve the thermosensitivity of a sec4-8 mutant of Saccharomyces cerevisiae. Therefore, it is proposed that RYL1 is a functional homologue of the S. cerevisiae SEC4 gene and is involved in the fusion of secretory vesicles with the plasma membrane in the general protein secretion pathway. Key words: Yeast 9 Yarrowia lipolytica 9 Protein secretion - Rab protein

Introduction The Rab protein family is a subdivision of the Ras protein superfamily initially described in a rat brain cDNA library by Touchot et al. (1987). It consists of tightly related small GTP-binding proteins sharing common features (for reviews, see Bourne et al. 1990; Grand and Owen 1991; Valencia et al. 1991), including a size of about 21 kDa, conserved blocks involved in GTP binding, and two cysteines at the C-terminal end which are post-translationally modified to interact with membranes (Rossi et al. 1991). This Rab family is widespread among eukaryotes and is implicated in a variety of membrane traffic processes (Rothman B. Pertuiset 9J.-M. Beckerich (~) 9C. Gaillardin Laboratoire de G6n6tique Mol6culaire et Cellulaire, INRA-CNRS, Institut National Agronomique, F-78850 Thiverval-Grignon, France Communicated by K. Esser

and Orci 1992) from the endocytic pathway (rab5) to the transport of synaptic microvesicles (rab6). This family could be subdivided in several groups of more tightly related sequences: the SEC4 subfamily, the YPT1 subfamily, and the RAB4 subfamily. It is supposed that these subfamilies share functional similarity. This hypothesis was supported by experiments of heterologous complementation and by intracellular localization in common compartments. For instance, YPT2 of Schizosaccharomyces pombe was able to complement a sec4 mutant of Saccharomyces cerevisiae (Haubruck et al. 1990), and RAB1 from mouse cells was able to complement the YPT1 deficiency in S. cerevisiae (Haubruck et al. 1989). At least, two components of the protein secretion pathway were shown to belong to this protein family in the yeast S. cerevisiae: Sec4p is involved in the targeting of the secretory vesicles to the plasma membrane (Goud et al. 1988; Walworth et al. 1989); Yptlp is implicated in the fusion of endoplasmic reticulum-derived microvesicles with the Golgi apparatus (Segev et al. 1988; Bacon et al. 1989). The site of action and the function of these two Rab proteins were assessed on conditional mutants by a combination of electron microscopy, immunofluorescence microscopy and cell fractionation. These studies showed that these proteins could control the vectorial flow of the vesicular transport process according to the model of Bourne (1988). The Rab proteins are associated with the cytoplasmic side of the carrier vesicle. In their GTP-bound form, they may interact with a receptor on the cytoplasmic side of the downstream compartment, thus targeting the vesicle. GTP hydrolysis allows recycling to the upstream compartment. Interaction with this compartment triggers nucleotide exchange from GDP to GTR It is thought that every vesicle fusion step is controlled by such a mechanism through different types of GTP-binding proteins like members of the Rab or the Arf subfamily (Rothman and Orci 1992). In order to identify genetic markers along the protein secretion pathway of the yeast Yarrowia lipolytica, a screening for members of the Rab family was undertaken. Four sequences called RYL (for Rab-related gene in Y. lip-

124

olytica) were selected and a m o n g them RYL1 was shown to be closely related to SEC4/YPT1. This gene is essential and a thorough sequence analysis led us to postulate that R y l l p could be the h o m o l o g u e of Sec4p. In order to confirm this hypothesis, the RYL1 coding sequence was placed under the control of the GALIO promoter in a S. cerevisiae expression vector and was shown to specifically complem e n t the sec4-8 growth defect.

Materials and methods Strains, media, and general molecular biology techniques. Standard media were used to grow Escherichia coIi, S. cerevisiae and Y. lipolytica (Sherman et al. 1983; Sambrook et al. 1989). The strains and plasmids used in this work are listed in Table 1.5-FOA selection was performed on minimal medium (1% glucose, 0.17% Difco yeast nitrogen base without ammonium sulphate, 0.1% proline as nitrogen source, supplemented as required) containing 10 rag/1 of uracil and 1.25 g/1 of 5-fluoroorotic acid (5-FOA; PCR Inc., Gainesville, Florida, USA). YPgal was 1% yeast extract, 1% peptone, 2% galactose. All restrictions, ligations and hybridization experiments were performed according to standard protocols (Sambrook et al. 1989). Labelled compounds were supplied by Amersham-France. Transformation techniques for Y. lipoIytica have already been described (Beeke-

rich et al. 1994). Plasmid stability tests were done as described by Fournier et al. (1991).

Plasmid construction. The inserts from the Y. lipolytica gene library were subcloned as AccI fragments in the SmaI site from the Bluescript SKi- plasmid (Stratagene). Plasmids pINA609 and pINA610 are Bluescript derivatives carrying RYL1 cloned in the two possible orientations with regard to the polylinker. To construct the disruption vector pINA845, pINA610 was partially digested by SphI and completely with NcoI, thus excising 121 bp upstream of the ATG of RYL1 and 250 bp of the coding sequence, and ligated to a 1.65-kb NcoI-SphI fragment carrying the URA3 gene marker from pINA300. Alternatively, the same digest of pINA610 was blunted with T 4 DNA polymerase and re-circularized on itself, to generate the deleted vector pINA847. To construct the replicative plasmids pINA841 and pINA844, carrying RYL1 and the LEU2 or URA3 gene markers, a 2.1-kb BamHI-ClaI fragment from pINA610 was cloned in the replicative vectors pINA237 and pINA398 cut by BamHI and ClaI, respectively. Use of the S. cerevisiae expression plasmid Yep52' has been reported previously (Beckerich et al. 1994). First, a 1-kb XbaIXbaI fragment carrying the whole RYL1 coding sequence starting 9 bp upstream of the initiator ATG was gel purified and inserted in the correct orientation in YEp52' opened by XbaI, downstream from the GALIO promoter. Second the LEU2 gene marker of YEp52' was substituted by URA3: a CIaI-FspI URA3 fragment from YIp5 was inserted in YEp52' opened by ClaI and EcoRV in the LEU2 region. The resulting plasmid pINA871 carrying the URA3 gene marker and RYL1 was used to transform the Sec Ura- mutants. A control plas-

Table 1 List of strains and plasmids Strain/plasmid

Genotype/description

Source

E. coli HB101 TG1 CJ236

hsdR-, hsdM-, recA13, SupE44, IacZ4, leuB6, proA2, thi-1, Sm R F-, recA1, endA1, gyrA96, thi, hsdR17 (rk, m~), supE44, relAl dut-1, ung-1, thi-1, relA-1; pCJ105 (Cmr)

B. Bachmann Stratagene Stratagene

135995

Reference n i wild-type a r t s MatA, lyslI-23, ura3-302, xpr2-322, leu2-270 MatB, hisl-1, ura3-302, Ieu2-270 MatA, lysll-23, ura3-302, xpr2-322, leu2-270, ryll : : URA3, [pINA841] MatA, lysl l-23, ura3-302, xpr2-322, leu2-270, ryll delta, [pINA844]

Our collection Our collection Our collection This work This work

S. cerevisiae NY405 Secl Secl2

Mata, ura3-52, sec4-8 Mat~, ura3-52, secl-1, leu2-3-I12, SUC2 Mata, ura3-52, secl2-1, leu2-3-112, his314, trpl-2891

B. Goud et al. (1988) R. Schekman R. Schekman

pBR322 with LEU2 of Y. Iipolytica and ARS18 inserted in the EcoRI site pBR322 with URA3 of Y. lipolytica inserted in Sall pBR322 with URA3 of Y. Iipolytica and ARS18 Initial RYL4 plasmid derived from pINA62 Initital RYL3 plasmid derived from plNA62 Initial RYL2 plasmid derived from pINA62 Initial RYL1 plasmid derived from pINA62 2.1-kb AccI fragment from plNA600 cloned at the Sinai site of Bluescript z~ 2.1-kb AccI fragment from pINA600 cloned at the Sinai site of Bluescript TM 2.1-kb ClaI-BamHI fragment of pINA610 inserted in pINA237 2.1-kb ClaI-BamHI fragment of pINA610 inserted in pINA398 URA3 fragment inserted into SphI-NcoI of pINA610 disrupting the RYL1 gene SphI-NcoI deletion of pINA610 Derivative of YEp52 with the polylinker of M13 tgl31 downstream from the GALIO promoter RYL1 inserted in XbaI-XbaI of YEp52', the insertion of URA3 in the ClaI-EcoRV deletion BamHI-BamHI deletion of RYL1 gene in plNA871

AM Ribet (personal communication) R Fournier (personal communication) R Fournier (personal communication) This work This work This work This work This work This work This work This work This work This work R Durrens (personal communication)

Y. lipolytica W29 E129 E146 2 - 1 1 1

Plasmids pINA237 pINA300 pINA398 pINA591 pINA594 pINA596 plNA600 pINA609 pINA610 pINA841 p!NA844 pINA845 pINA847 YEp52' pINA871 pINA872

This work This work

125 mid, plNA872, was constructed by deleting a 1-kb BamHI fragment carrying the whole RYL1 coding sequence from pINA871.

der to screen specifically for members o f the S E C 4 and YPT1 subfamilies, stretches of similarity were searched between several Rab-related protein sequences with the Screening of the gene library. A genomic DNA library of the Y. lipolytica reference strain W29 (Xuan et al. 1988) was screened by col- C L U S T A L alignment program. The sequence T V K L Q I ony hybridization. Bacterial colonies were replicated and hybridized W D T A G Q E R F R T I T (single amino-acid code) was conon nitrocellulose filters (Sambrook et al. 1989). The 57-met oligo- served in S. cerevisiae Y p t l p and related S. p o m b e gene nucleotide TVK (see Fig. 1 A) was synthesized on a Biosearch Cy- products, it is also the longest stretch of similarity between clone DNA synthesizer and purified by polyacrylamide-gel electrophoresis. It was labeled at its 5 terminus with 32p by T4 polynucle- members of the YPT1 and S E C 4 subfamilies. An oligonuotide kinase and used as a probe. Hybridization was carried out at cleotide sequence (TVK, see Fig. 1 A) was deduced from 47~ for 16 h in a 7% SDS, 1 M sodium phosphate buffer. Filters the amino-acid sequence using the codon bias of a set of were dipped in lxSSC (150 mM NaC1, 15 mM Na citrate), washed Y lipolytica genes including XPR2, LEU2, P H 0 2 , and three times in 0.1xSSC, 10% formamide, 0.1% SDS at 47 ~ for 20 LYS5 (Davidow et al. 1987a, b; Xuan et al. 1990; Treton rain and rinsed in txSSC before autoradiography. et al. 1992). This strategy implicated a high probability of Sequence analysis. Restriction fragments were subcloned in the poly- mismatches due either to a different codon usage or to Iinker of Bluescript TMplasmids (Stratagene) and sequence analysis amino-acid substitutions. To obviate these possibilities, a was performed by the dideoxy chain-termination method on singleor double-stranded plasmid using T7-modified DNA polymerase long oligonucleotide was chosen in order to give clear sig(Sequenase, US Biochemicals). The sequencing reactions were nals despite some mismatches under relatively stringent primed either with commercial primers supplied by Stratagene or hybridization conditions. This oligonucleotide was used to with internal primers synthesized on a Cyclone Biosearch synthetis- screen a Y lipolytica gene library by colony hybridization er and purified by polyacrylamide-gel electrophoresis. The sequenc- (see Materials and methods). Fifteen positive clones were es of RYL1 and RYL2 were registered in the GENBANK database under the accession numbers L06969 and L06970, respectively. The grouped in four classes by restriction analysis and hybridsequence data were analyzed by using the computing facilities of- ization, and each type of insert was sequenced. The sefered by the "Base Informatique sur les S6quences d'Acides quences were translated and compared with sequences Nucl6iques pour les Chercheurs Europdens" (BISANCE) at CITI2- available in databases using the FastA computer program Paris (Dessen et al. 1990) and with the UWGCG package (Devereux (Pearson and L i p m a n 1988). Four different Ras-type genes et al. 1984). were identified and named RYL1 to RYL4 (Ras-related gene Plasmid shuffling. Mutations were generated in pINA610 by the pro- from Y lipolytica). The matches of these sequences with cedure of Kunkel et al. (1987), with a kit supplied by BioRad Labor- the T V K oligonucleotide are shown on Fig. 1 A. atories (Hercules, USA) and following the manufacturer's instrucF r o m the FastA data, R y l l p appeared to be tightly retions. The oligonucleotides used for the G139D mutation and for the N124I mutation were ATCTGCCAAGGCCTGGTCCTGTTCTGT- lated to the Sec4p subfamily of Rabs and Ryl2P to Rab5p GGAAAC and ATCGAGGTCGCACTTGATGCCAACCAGAAT- or Rab2p. Ryl3p was highly h o m o l o g o u s to the Cdc42p GAG (mutations are in bold). The mutated DNAs were screened by subfamily and Ryl4p to the Rho subfamily (data not sequencing for the presence of the mutations and transferred as Bam- shown). The Cdc42 and Rho subfamilies are distinct subHI-ClaI fragments to the replicative vector pINA237 (carrying the LEU2 marker) opened by BamHI and ClaI. The Leu- strain divisions of the large Ras superfamily and their members 135995, carrying a deletion of RYLI complemented by the replica- regulate respectively cell growth (Johnson and Pringle rive plasmid pINA844 (carrying the URA3 marker), was construct- 1990) and cytoskeleton organisation (Ridley and Hall ed as follows. A t.7-kb BamHI-ClaI fragment of pINA847 was used 1992). As RYL1 corresponded to our initial goal, we foto transform strain 111-27 (see Table 1), so as to replace the ryll : : URA3 insertion present in this strain by a SphI-NcoI deletion of RYL1 cused our studies on it. The RYL1 coding sequence was 203 amino acids long (see Fig. 4). The cells were screened on 5-fluoroorotate to select for Ura- transformants (Boeke et al. 1987). Six Ura- clones were ana- and 617 base pairs upstream o f the initiator codon were delysed by Southern blots of total DNA digested by Asp718 and PstI. termined (see Fig. 1 B). H o m o l o g y relationships were anFive of them displayed the pattern expected for the substitution event. It was now possible to exchange the resident Leu + Ryl+ plasmid alyzed by three approaches. As shown on Fig. l B and 2, (plNA841) for a Ura + Ryll + plasmid (pINA844) (see Table 1), so as RYL1 showed typical features o f a Ras-like protein (Valento obtain strain 135995. This strain was transformed by the mutated cia et al. 1991), including conserved motives G1, T43 and plasmids, selecting for Leu + transformants. Shuffling of the plasmids G2 (phosphate/magnesium binding) and F36, G3 and G4 was performed on 5-fluoroorotic acid to counterselect the resident (guanine base-binding); there were two cysteines in a CpINA844, and the phenotype of the Ura- Leu+ segregants was tested at different temperatures. The replicative plasmids were re-ex- terminal position which m a y allow m e m b r a n e interaction tracted from the yeast cells, and the entire coding sequence was through geranylgeranylation (Rossi et al. 1991). As exchecked by sequencing with a set of oligonucleotides to verify the pected for a Rab protein, the sequence conservation was presence of the induced mutation and the absence of unwanted sec- very high around the G3 position whereas the C-terminal ondary mutations. end downstream from G4 was hypervariable. A multiple alignment program (Pileup from U W G C G ) on the entire polypeptide sequences showed (Fig. 3) that R y l l p was Results tightly related to the Sec4p subfamily and distinct from the Y p t l p subfamily and from other Rabs. To confirm this hyCloning o f Rab-related genes pothesis, the L7 regions of several Rabs were compared with a multiple alignment program. Divergence was obAs emphasized in the introduction, the Rab gene products served between the Rab members at the level of the L7 share large blocks of fully conserved amino acids. In or- loop. This region corresponds to the L7 loop of p21 ras

126 A

TVK

GGTGATGGTTCGGAATCGCTCCTGTCCGGCGGTGTCCCAGATCTGG~GCTTGACGGT

RYLI

GGTGATGGTTCGGAAcCGCTCCTGgCCGGCcGTGTCCCAcActTGcAGCTTGACr

RYL2

GGccATGcTTCGGAATCGCTCCTGcCCcGCcGTGTCCCAtATtTGcAGCcgcACcGT

RYL3

cGcagTcGGTCGtAATC.CTCCTGaCCGGCGGTGTCaaAcAgtccgAtgTgtAgGGc

RYL4

cGcagTcGgTCGtAATC.CTCCTGgCCGGCGGTaTCCCAcAgggccAGCTccACtCg

A A C T A T A T A T C A A G T C T A T T A A T C A G T T T G G • G G C T C G T C C T C C A C T A T C G T C T C C C T • C G T • A T G T C C G G T A G C A T G • C A C C A G C C C C T G C T A A T A G A G - 101 T T C A C A A T C A C A G G A C T A T C T C C C T GCG C A C C A C G A G A G A A G T C C A C C A T C A T C T T T T G A A C A C A C A C C A A C A A C A C A C C T T CGTGATTGTCTAGATAC C

-I

M S H S Q R T Y D L L I K L L L I G D S G V G K S A T G T C G CAC T C C C A G A G A A C A T A C GAC CTG CTG A T C AAG CTG CTG CTG A T A GGC GAC TCC GGC GTG GGC AAG TCG

75

26

C L L L R F C E D Q F T P S F I T T I G I D F K T G T CTG CTG CTG C G G TTC TGC GAG GAC CAG T T C A C C CCC TCC TTC A T C ACC A C C A T T GGT ATC GAC T T C A A A ATC

150

51

R T I D I G N Q R V K L Q V W D T A G Q E R ,F R T C G A A C G ATT GAC A T T GGC A A T CAG CGG GTC AAG C T G CAA GTG TGG GAC ACG GCC GGC CAG GAG CGG TTC CGA ACC

225

i T T A Y Y R G A }4 G I L L V Y D V T D E K S F N A T C A C C ACC GCC T A C T A C CGG GGC GCC A T G GGC A T T CTG CTG GTG T A C GAC GTG A C G GAC GAA AAG TCC T T C AAC

300

1

76

I01

N

I

E

N

W

Y

Q

N

V

Q

T

Y

A

N

E

G

V

E

L

I

L

V

G

A A C A F T GAA A A C T G G TAC CAG A A C GTG CAG A C G T A C GCC AAC GAG GGC GTC GAG CTC ATT CTG GTT GGC

126

151

176

201

C

D

L

D

E

~

R

V

V

S

T

E

Q

IG~I Q

A

L

A

D

K

F

G

I

375

P

F

T G C GAC CTC GAT GAG A A G C G A GTC GTT TCC A C A G A A CAG GGC CAG GCC TTG G C A CAT AAG TTT GGC A T T CCC TTC

450

L E A S $ K T N I N V E E C F u $ V A T R I R D T T T G GAG GCC T C A TCC A A G A C C AAC A F T A A C GTC G A G GAG TGT TTC T A C TCG GTT GCC ACT C G A A T C A G G GAC ACT

525

V A K T K G N E S G $ G G I N I A E G E E N $ A $ GTG GCC AAG A C C A A G GGC A A C GAG TCT GGG T C G GGC GGC ATC AAC A T C GCC GAG GGC GAG G A A AAC TCC GCC TCC

600

K C C A A G TGC TGT T A A C T G A A G A G G G T A T A T A G T A T A T T A A T T G • G T C A T G A A T T A T A G T A A G A T G • C A G T C A G A C T C T G T G A T G T A G T G T T G T A A G T A T

696

CTACCAGTAGTACAATGGTTGGTTTCGCTTTCTTGTTAG G A C A C A C T T C A G G A C T A T C G A C C T A C T T C T A C A T C T T T A G A T A A A T A C A A A C G A T T C C ~ G

796

T T C T C T A T G G C A G T A C T G T G A A A G A T A A A T C A A C A TG T T A G T A T C A T A A T G G A G A A G T GTC G A T A C A C T T T A A C T A G C T A C T T G T A C T G G T A C T T C T A G C

896

T A C A G T A A C T G G T G G A A C C G C C C GTT C T T G T G C T G G T A C T C G T A C T G G T A C A C T T G T A

954

Fig. 1 A alignment of the TVK probe with the cloned sequences. The sequence of the TVK oligonucleotide is aligned with the matching sequences in the four cloned genes, Matching bases are in boldface capital letters; non-matching bases are in lower-case letters; dots indicate gaps in the matching area. B sequence of RYL1. The nucleotide sequence is numbered on the right and the translation of the open reading frame is numbered on the left. The sequence hybridizing to the TVK oligonucleotide is underlined. The position and the nature of the induced mutations are boxed. This sequence is registered in GENBANK under the accession number L06969

which may control the specificity of these proteins since Dunn et al. (1993) and Brennwald and Novick (1993) showed that it was able to confer a Yptlp-like function on Sec4p. The relationships between the Rab polypeptides were not modified when either the L7 regions or the entire sequences were aligned (data not shown) and R y l l p was here also a homologue of Sec4p. Therefore, we concluded that Ryllp belonged to the Sec4p subfamily.

RYL1 is an essential gene Two strategies were adopted to disrupt the RYL1 coding sequence. (1) The RYL1 coding sequence was disrupted by a URA3 fragment in a diploid strain and the progeny of this strain was studied: viable Ura § clones in the progeny would indicate that disruption of RYL1 was compatible with spore germination and vegetative growth. (2) The same disruption was performed in a haploid strain containing a wildtype RYL1 gene on a centromeric plasmid: loss of this plasmid would again indicate that disruption of R Y L I did not prevent vegetative growth: The diploid strain U r a - L e u - E129/E146 was transformed with a 3.4-kb B a m H I - ClaI fragment from pINA845 (see Materials and methods and Fig. 4) selecting for Ura § clones, and the disruption of one of the RYL1 alleles was confirmed by Southern blots on several transformants. One of these diploids was sporulated and the progeny was studied by random spore analysis: among 110 auxotrophic seg-

7 1 Sc-YPTI Hu-RABI Sp-YPT2 Dd-SASI Y1-RYLI Sc-SEC4 Hu-RAB4 consensus

50

............ MNSEYDYL FKLLLIGNSG ......... M SSMNPEYDYL FKLLLIGDSG ........... MSTKSYDYL IKLLLIGDSG ..... M T S P A T N K S A A Y D Y L I K L L L I G D S G ......... M SHSQRTYDLL IKLLLIGDSG MSGLRTVSAS SGNGKSYDSI MKILLIGDSG ............ MSETYDFL FKFLVIGNAG ................ YDYL -KLLLIGDSG (

VGKSCLLLRF VGKSCLLLRF VGKSCLLLRF VGKSCLLLRF VGKSCLLLRF VGKSCLLVRF TGKSCLLHQF VGKSCLLLRF )

51 STIGVDFKIK STIGVDFKIR TTIGIDFKIR TTIGIDFKIR TTIGIDFKIR TTIGIDFKIK HTIG~/EFGSK -TIGIDFKI)

TVELDGKTVK TIELDGKTIK TIELDGKRIK TIELEGKRIK TIDIGNQRVK TVDINGKN'VK IINVGGKYVK TIE--GK-VK

LQIWDTAGQE LQIWDTAGQE LQIWDTAGQE LQIWDTAGQE LQVWDTAGQE LQLWDTAGQE LQIWDTAGQE LQIWDTAGQE <

Effector

Sc-YPTI Hu-RABI Sp-YPT2 Dd-SASI YI-R~LI So-SEC4 Hu-RAB4 consensus

i01 DVTDQESFNG DVTDQESFNN DVTDKKSFDN D%"PDEKSFGN DVTDEKWFNN DVTDERTFTN DITSRETYNA DVTD---F-N

RFRTITSSYY RFRTITSSYY RFRTITTAYY RFRTITTAYY RFRTITTAYY RFRTITTAYY RFRSVTRSYY RFRTIT--YY )

Hu-Rabl

i_

Sp-Yptl Sc-Yptl

i00 RGSHGIIIVY RGAHGIIVVY RGAMGILLLY RGAMGILLVY RGAMGILLVY RGAMGIILVY RGAAGALLVY RGA-GI-LVY

Dd-Sas2

t

G2

VK~KgLQEIDR %rKQWLQEIDR VRTWFSNVEQ IRNWIRNIEQ IENWYQNVQT IKQWFKTIrNE LTNWLTDILRM ---WL ......

YATSTVLKLL YASENVNKLL HASENV'fKIL HATDSVNKML YANEGVELIL HANDEAQLLL LASQNIVIIL A-E-V---L (

VGNKCDLKDK VGNKCDLTTK IGNKCDCEDQ IGNKCDMAEK VGNKCDLDEK VGNKSDM.ET CGNKKDLDAD VGNKCDL-E) G3 )

150 RWEYDVAKE KVVDYTTAKE RQVSFEQGQA KVVDSSRGKS RVVSTEQGQA RVVTADQGEA REVTFLEASR RW .......

Sc-YPTI Hu~RABI Sp-YPT2 Dd-SASI YI-RYLI Sc-SEC4 Hu-P.AB4 Consensus

151 FADANKNPFL FADSLGIPFL LADELGVKFL LADEYGIKFL LADKFGIPFL LAKELGIPFI FAQENELMFL LAD-LGI-FL

Sc-YPTI Hu-RABI Sp-YPT2 Dd-SASI Y1-RYLI Sc-SEC4 Hu-RAB4 Consensus

201 QKKEDKGNVN LKGQSLTNTG E K S .... N V K I Q S T P V K Q S G FSNQAN.NVD LG.NDRTVK. QPQVVQPGTN LGANNNKKK. ESGSGGINIA EGEENSASK. NGKEGNISIN SGSGNSSKS. YGDAALRQLR SPRRTQAPNA ........ V .............

ETSALDSTNV ETSAKNATNV EASAKTNVNV ETSAKNSINV EASSKTNINV ESSAKNDDNV ETSALTGEDV E-SAK---NV ( > S4

Dd-Sasl At-Ara3 Sp-Ypt2 Do-Rab8 Do-Rabl0

__~

L7

Sc-Sec4 YI-Ryll

2OO EDAFLTMARQ EQSFMTMAAE DEAFFTLARE EEAFISLAKD EECFYSVATR NEIFFTLAKL EEAFVQCARK EE-F--LA--

225 GGC.C GGC.C .RC.C .AC.C ..C.C .NC.C QECGC C-C

1

Vc-Yptvl

SDDTYTNDYI ADDTYTESYI SEDSFTPSFI SEDSFTPSFI CEDQFTPSFI VEDKFNPSFI IEKKFKDDSN -ED-FT-SFI ( --

G1

Sc-u Hu-RABI Sp-YPT2 Dd-SASl YI-RYLI Sc-SEC4 Hu-RAB4 consensus

2

I K Q S N S Q Q N L ...... N E T T I K K R M G P G A T ...... A G G A Fig. margo3rdneD e f h sp oihsnoti talern e e wp t el b ld y - R n m ae m I K K Q K I D A E N ...... E... s r e fe b .hb y aol sti mrai f hmargTordnesd a de t curwt snoce y h b t "PileI K K R M I D T P N ...... E... mN... a r gmoup"rop r G CfgG W n U ei es hu ms g n at i ts t es sn a i Fig. 1 A. I R D T V A K T K G ...... I Q E K I D S N K L ...... e bVhG VsG naiTe t d Roerdpu l sc nni i hnois i trap emoc r d eed n ew t xoge o t D ILNKIESGEL DPERM8 G S G I Qb d a, 0nR l ba oa R t d YptV1, no a t I ...................

(Do) dopsis3 thaliana - a (At) r A

9

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Fig. 4 A, B Construction of chromosomal deletions at the RYL1 locus. Chromosomal sequences at the RYL1 locus are indicated by open boxes, the URA3 sequence by a black box, and vector sequences by a line. The arrow indicates the position of the RYL1 open reading frame; the dashed part of the arrow indicates the part of the ORF which is deleted. A disruption of RYL1 by the URA3 gene: a BamHI-ClaI fragment from pINA845 was used to disrupt the chromosomal RYL1 gene by a double cross-over (dotted line). B replacement of the URA3 disruption of RYL1 by a deleted RYL1. This exchange was performed in the 111-27 strain carrying the replicative RYL1 + plasmid pINA841 (data not shown) to ensure viability. A 1.7-kb BamHI-CIaI fragment of pINA847 was used to replace the URA3 disruption by a SphI-NcoI deletion (see text) by a double cross-over

Fig. 5 A map of the plasmid pINA871. The RYL1 coding sequence was placed downstream from the GALIO promoter and the URA3 gene marker was inserted in place of LEU2 as described in Materials and methods. The control plasmid pINA872 was obtained by deleting the BamHI-BarnHI fragment carrying the entire RYL1 coding sequence. B growth of strains sec4-8, secl-1 and sec12-1 transformed by pINA871 (RYL § or pINA872 (RYL-). Growth on YP gal at 30 ~ and 37 ~ is presented

Site-directed mutagenesis

RYL1 complements a thermosensitive sec4- mutation

In order to study the function of Ryllp, we attempted to obtain conditional mutants. In S. cerevisiae sec4-8 mutants, the amino-acid G147D change leads to thermosensitivity (Salminen and Novick 1987). The corresponding mutation (G139D) was made in pINA841. Strain 135995 carrying the ARYL1 mutation and pINA844 was transformed by the mutated plasmid, and plasmid shuffling was attempted (see Materials and methods). It was impossible to cure the resident Ura + Ryll § plasmid at any temperature. It was thus concluded that the mutation G139D conferred a lethal phenotype in Y.. IipoIytica. As an alternative, the substitution N124I was induced. From studies on S E C 4 and YPT[7 in S. cerevisiae and H-ras in human cells (Walworth et al. 1989), this mutation should lead to a dominant lethal phenotype. Plasmid shuffling was attempted as previously. Quite surprisingly, the resident Ryl + plasmid could be lost in this case and the phenotype of N124I was wild-type.

As discussed above, the R Y L I gene seemed to be a Y. lipolytica homologue of SEC4 from S. cerevisiae. In order to sustain this hypothesis, the RYL1 gene was expressed in S. cerevisiae and its ability to complement a thermosensitive sec4-1 mutation was tested. To this end, the RYL1 gene was inserted downstream from the GALIO promoter in plNA871 (Fig. 5 A) and as a control pINA872 was obtained by deleting RYL1 from pINA871. The NY405 strain was transformed with pINA871 and pINA872. The transformants were studied by drop tests on different growth media at different temperatures (see Fig. 5 B). Growth was observed at 37 ~ only with pINA871 transformants and on galactose-induction media. This growth property was unstable and segregated with the plasmid. Under the same conditions, pINA872 supported growth at 30 ~ but not at 37 ~ It was concluded that growth at 37 ~ was conferred by complementation of the S E C 4 gene defect by RYL1 (Fig. 5 B).

129 A simple duplication of SEC4 was shown by Salminen etal. (1987) to suppress several thermosensitive sec mutations in S. cerevisiae: secl5-1 is fully suppressed, sec2-41 and sec8-9 are partially suppressed (strains grow at 33.5 ~ as are secl-1, sec5-24, seclO-2 and sec19-1 though to an even more limited extent. With the exception of sec19-1, which is not well assigned in the protein secretion pathway, these mutations affect the same late segment of the protein secretion pathway. To assess the specificity of the suppression of sec4-1 by RYL1 and to test the hypothesis of a by-pass, the thermosensitive mutants secl-1 and sec12-1 (see Table 1) were transformed with pINA871. sec]-i was chosen as an example of a mutant partially suppressed by SEC4 and impaired at the same step of the secretion pathway as SEC4. sec12-1 was chosen as an example of a SEC4 non-suppressible mutant impaired at an earlier step of the pathway. No growth was observed at 37 ~ (Fig. 5 B) indicating that suppression of sec4-1 by RYL1 was specific.

Discussion In this paper, we describe the isolation of RYL1, a SEC4 homologue, in the yeast Y. lipolytica. Two lines of evidence indicate that RYL1 is a functional homologue of SEC4. First, the sequence of Ryllp appears to be tightly related to members of the Sec4p subfamily, especially to Ypt2p of S. pombe. Second, as in the case of YPT2 (Haubruck et al. 1990), RYL1 was shown to complement the sec4-8 growth defect when expressed in S. cerevisiae. This complementation was specific since RYL1 did not suppress secl-1, which is partly suppressed by SEC4 § (Salminen and Novick 1987), nor sec12-1 which is not suppressed by SEC4 § but by another Ras-related gene, SAR1 (Nakano and Muramatsu 1989). Work on complementation by chimeric YPT1/SEC4 constructions showed that it was more difficult to complement sec4-8 than yptl- 1 (Brennwald and Novick 1993; Dunn et al. 1993). Taken together, these results strongly suggest the functional homology of RYL1 and SEC4. In order to study the function of RYL1, directed mutagenesis was undertaken using the data accumulated on Rab genes. The homologue of the thermosensitive sec4-8 mutation appears to be fully lethal in RYL1, confirming that this position is critical for the function of Sec4p. More surprising is the phenotype of the mutation N124I, homologous to N121I in YPT1. The Asn residue affected is localized in the G3 block and is involved in the interaction with the guanine ring of the bound nucleotide. This position is conserved across the whole Ras superfamily and its change to Ile leads to a dominant lethal phenotype in YPT1 ile121 (Wagner et al. 1987), to a dose-dependent lethal phenotype in SEC4 iI~ (Walworth et al. 1989), or to a transforming phenotype in the H-ras ile116 proto-oncogene (Barbacid 1987). In RYL1, this mutation displays a wild-type phenotype when expressed on a centromeric vector. Although no definite explanation of this result can be proposed at this

stage, it may be noted that intragenic suppressors of yDtl ile121 in S. cerevisiae were found at several positions along the sequence (Schmitt et al. 1988), so that interactions between variable region(s) of RYL1 and the mutated G3 block could account for our results. The production of conditional mutants will be a prerequisite to the study of the function of RYL1 in Y. lipolytica. Acknowledgements We thank M. Aigle for the gift of the GALIO expression vector. This work was supported by the Institut de la Recherche Agronomique, by the Centre National de la Recherche Scientifique and by an EEC grant (BIOT-CT91-0267DSCN).

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