Gene, 99 (1991) 39-46

39

Elsevier

GENE

03916

Structural and functional conservation between the high-affinity uvarum and Saccharomyces cerevisiae (Ion transporters; enrichment)

transmembrane

domains;

hybridization

cloning;

recombinant

K+ transporters of Saccharomyces

DNA;

inter-species

comparisons;

size

Julie A. Anderson, Laura A. Best and Richard F. Gaber Department of Biochemistry, Molecular and Cell Biology, Northwestern University,Evanston, IL 60208 (U.S.A.) Received by J.A. Gorman: 24 July 1990 Revised: 9 October 1990 Accepted: 10 October 1990

SUMMARY

In Saccharomyces cerevisiae, high-affinity K + uptake is dependent upon a 180-kDa plasma membrane protein encoded by TRKI (c-TRKI) [Gaber et al., Mol. Cell. Biol. 8 (1988) 2848-2859)]. Although hybridization with a c-TRKl probe revealed highly homologous sequences in the genomes of most Saccharomyces species, the TRKI sequence in S. uvarum (u-TRKl ) was detected only under low-stringency conditions. We cloned u-TRKI and found it to confer high-affinity K + uptake in S. cerevisiae. A comparison of the inferred amino acid sequences reveals 78 y0 identity and 86 y0 similarity between the two high-affinity transporters. The most highly conserved regions are the putative membrane-spanning domains (95% identical), suggesting that the structure of the transmembrane domains is important for high-afinity K’ transport.

1987;

INTRODUCTION

The structure-function relationship of ion-transporting proteins remains poorly understood, despite knowledge of the primary structures of a number of eukaryotic ion transporters (Noda et al., 1984; Kawakami et al., 1985; Shull et al., 1985; 1986; Briggs et al., 1986; Kent et al., 1987; Numa, 1987; Gunteski-Hamblin et al., 1988; Rudolph et al., 1989). In only two cases, that of the photosynthetic reaction centers from Rhodopseudomonas viridis (Deisenhofer et al., 1985) and Rhodobacter sphaeroides (Allen et al.,

Correspondence to: Dr. R.F. Gaber, 60208 (U.S.A.) Abbreviations: d,

deletion;

2153 Sheridan

Road, Evanston,

IL

Tel. (708)491-5452. aa, amino GABA,

acid(s);

y-amino

bp, base

butyric

pair(s);

acid;

kb,

c, S. cereviriue; kilobase(

K,,

Michaelis-Menten constant; LS, low salt; M, membrane-spanning domain; nt, nucleotide(s); ORF, open reading frame; PEST, Pro Glu Ser Thr; S., Saccharomyces; SSC, 1.5 M NaCI/O.lS

M Na, ‘citrate

SDS, sodium dodecyl

of K + ; u, S. uvarum; [ 1,

denotes

plasmid-carrier

0378-I 119/91/$03.50

0

sulfate; TRK, transporter state.

1991 El sevier Science Publishers

B.V.

pH 7.6;

Yeates et al., 1987; 1988), have high-resolution crystal structures of integral membrane proteins been determined. Although three-dimensional structural analysis can greatly facilitate an understanding of the molecular basis of protein function, the difficulty of crystallizing membrane proteins has resulted in a lack of such information. We are pursuing molecular genetic approaches to study the structure and function of K’ transporters in S. cerevisiae. The isolation of K’ transport-deficient mutants (Rodriguez-Navarro and Ramos, 1984; Gaber et al., 1988; Ko et al., 1990) allowed us to clone the genes likely to encode the high-affinity (Gaber et al., 1988) and lowaffinity (C.H. Ko and R.F.G., manuscript in preparation) K’ transporters from S. cerevisiae. Biochemical and molecular analysis of c-TRKl revealed that it encodes a 180-kDa membrane protein with twelve transmembrane domains, M 1-M 12, and a large, 650-aa hydrophilic region between M3 and M4 (Gaber et al., 1988). Further investigation of this transporter would be facilitated by knowledge of its topology within the lipid bilayer and the identification of domains essential for transport of K'

40

The nt sequences able to complement mutations in homologous genes of different species indicate inter-species conservation of functionally important regions. In Saccharumyces for example, centromeric DNA from S. uvarum and the STE2 gene from S. kiuyveris are functional in S. cerevisiae, due to the conservation of structural domains (Huberman et al., 1986; Marsh and Herskowitz, 1988). Deletion of TRKl in S. cerevisiae (trkld) results in the inability to grow on media containing low levels of K + . To identify specific regions of TRKl likely to be essential for ion transport, we cloned u-TRKl, showed that it confers high-affinity K + uptake in S. cerevisiae trkfd cells and compared its inferred aa sequence to c-TRKl.

Fig. 2. Detection

of TM/-related

species by Southern

analysis.

sequences

Genomic

romyces species by the mini-preparation (1987). Hind111 + EcoRI-digested 0.8% agarose RESULTS

AND DISCUSSION

[r-“P]dCTP

(a) Identification of TRKl in Sacchavomyces species A DNA fragment containing part of the coding region of c-TRKf (Fig. 1) was used as a probe to detect related DNA sequences in various yeasts. By Southern analysis, we failed to detect TRKl-related sequences in Candida utillis, Pichia modanensis, S. lipolytica and Schizosnccharomyces pombe (not shown). Similar analyses of genomic DNA from KluyveromJces lactis (not shown) and eight different Saccharomyces species detected a single TRKl-related sequence in each case (Fig. 2). In seven of eight Sacchffro-

from Sacchu-

method of Hoffman

and Winston

to nylon membrane

probe was prepared

fragment

from

primer

(Fig. 1) and method

1983; 1984). The filters were hybridized labeled probe and washed at 55°C (low stringency)

(Feinberg

overnight

to Kodak

et al.,

of the 1.3-kb

radiolabeled

with

and Vogelstein, at 65°C x

three times for 15 min in 6 and exposed

on a

(Maniatis

by gel purification

pRG387-1

by the random

~uce~u~~~7.~ce~

DNA (5 pg) was electrophoresed

gel and transferred

1982). The c-TRKI .YbuI-ClaI

in other

DNA was isolated

with the

SSCjO.1 Y; SDS

X-OMAT

AR film for

24 h.

myces species tested, the hybridization signals detected after high-stringency washing were equal in intensity to the S. cerevisiae signal, indicating a high degree of similarity between S. cerevisiae and the TRKI genes of these species.

pLB8-5 U-TRKI

-

C-TM

PUClB

Fig. 1. Comparison

of the restriction

maps of u-TRKl

pRG295-

1

pRG387-

1

I

and c-TRKZ. Plasmid

pRG295-1

contains

the full length clone of c-i’RK1 (Gaber

et al., 1988) and

pLBS-5 contains the u-TRKl clone. The black arrows indicate the length and direction of the u- and c-TRKZ ORFs. Plasmid pRG387-1 contains an XbaI fragment oft-TMl (Gaber et al., 1988). To clone u-TRKI, a Southern blot of S. urarnrn genomic DNA, digested with various restriction endonucleases. was probed with an XbaI-ClaI fragment from pRG387-1, indicated by the hatched box, and washed under low stringency conditions. Restriction digests were performed as described by Maniatis et al. (1982). Autoradiography revealed hybridization of the c-TRKI probe to a single 19-kb Hind111 fragment, a single IS-kb EcoRI fragment to enrich

for those

containing

or, when digested u-Tfiiyl

with both enzymes,

in the following

manner:

a single 8-kb fragment S. uv(1~111 genomic

(not shown).

DNA

DNA fragments

was digested

were sequentially

with HiEd

and,

following

get puritied agarose

electrophoresis, the 19-kb region was cut from the gel, the DNA eluted, and subsequently digested with EcoRI. The Hind111 + ~c~RI-digested was eiectrophoresed a second time and the 8-kb region was cut from the gel, the DNA eluted and ligated to HindHI + EcoRI-digested vector pRG415-2

(Gaber

S. uvarum genomic by the method The purified

et al., 1988). Plasmids

containing

DNA. To identify clones containing

of Grunstein plasmids

and Hogness

were retested

S. uvarunr inserts u-TRKl

(1975). Plasmid

for the presence

were cloned by transformation

sequences,

colonies of bacterial

DNA was prepared

of u-TRKl

from bacterial

DNA by Southern

blotting

of E. coli HBlOl

transformants

transformants and probing

generating

were screened as described

a partial

with the c-TRKl

gel

DNA DNA,

library

of

fragment

by Holmes and Quigley (1981).

with the c-TRKI

fragment.

41 Hybridization

to S. uvarum DNA,

much weaker

however,

signal, one that was removed

revealed

a

(c) Function of u-TRKl in Saccharomyces cerevisiae High-affinity K + uptake is abolished in S. cerevisiae trkld cells resulting in their inability to grow on media

by high-strin-

gency washing (not shown). These results suggested that greater evolutionary divergence had occurred between the TRKl gene of u-TRKI and those of the other Saccharomyces species. Consistent with this conclusion, the pattern of restriction endonuclease sites in and around u-TRKl was also different from that observed in the other species (Figs. 1 and 2). Sequence evolutionarily

containing low concentrations of K + (0.2 mM KCl) (Gaber et al., 1988). To determine if the cloned DNA fragment in pLB8-5 contained a functional gene capable of suppressing the trkld mutation in S. cerevisiae, the plasmid was introduced by transformation into a Ura- Trk- S. cerevisiae recipient (R1155; Table I). All Ura+ transformants acquired the ability to grow on medium

comparisons of functionally related but divergent transporters could provide infor-

containing

0.2 mM

KC1 (Fig. 3a).

mation on structurally important regions. To compare the two transporters, we cloned u-TRKl, tested its ability to mediate K + transport in S. cerevisiae and determined its nt

To demonstrate that u-TRKl confers increased K + uptake, S. cerevisiae trkld cells were transformed with centromeric plasmids containing either c-TRKl, u-TRKl or the YCp50 vector (R1262, R1745 and R1260, Table I) and were assayed for K’ uptake. The results of these assays demonstrated that u-TRKl and c-TRKl restore K + uptake in trkld cells (Fig. 3b). The affinity of u-TRK 1 for K + was determined in 86Rb + flux assays. The trkld cells containing YCp50 (R1260) exhibit a K, of approx. 12 mM whereas cells harboring c-TRKl (R1262) or u-TRKl (R1745) exhibit K,s of approx. 0.3 mM and 1 mM, respectively (Fig. 4). These results demonstrate that u-TRKl confers high-affinity K’ uptake to S. cerevisiae trkld cells.

sequence. (b) Cloning of u-TRKl from Saccharomyces uvarum Gene u-TRKI was cloned by sequentially gel-purifying genomic DNA fragments (Fig. 1). A library of the sizefractionated DNA fragments was screened by colony hybridization with the c-TRKI probe. This strategy proved to be very efficient; two positive clones were identified among 28 E. coli transformants. Plasmid DNA, designated pLB8-5 (Fig. l), was purified from one of these transformants for further analysis. Plasmid pLB8-5 was found to contain an 8-kb HindIII-EcoRI insert which hybridized to the c-TRKI probe. The hybridization signal was completely removed from the filter upon high-stringency washing (not shown).

(d) Sequence of u-TRKl The nt sequence of a 5-kb fragment containing the functional u-TRKl gene in pLB8-5 was determined (Fig. 5).

w

(a)

LS

IOOK

LS 0.2K

h-k/A [YCP501

lA [YcPSOl

tfk

trk

/A [u-TRK/l h-k/A

trkld

[u-TRKfl

[c-TRK/l

trkld

[c-TRKII

I I

0 Fig. 3. S. cerevisiue cells containing low levels of K + Approx. are described

onto LS (low salt) media lacking trklA[c-TRKI]

in Table I. YPD and YNB media and routine genetic techniques was made as previously

et al. (1983). (b) Ability oft-TRKl R1262 and R1745, respectively, et al., 1988). An arrow

indicates

20min

u-TRKI and c-TRKl tested for growth on low K + and net K + uptake. (Panel a) Test for growth on medium containing

1 x 10’ cells/ml were spotted

100 mM (left panel) and 0.2 mM (right panel) KCI. The rrkld[YCpSO], no K + (before its addition),

I

IO

and u-TRKl are described

described

(Gaber

Ura (to maintain

selection

and trklA[u-TRKZ] strains

were as described

by Sherman

et al., 1988). Yeast transformation

in Table I. Extracellular addition

K’

was measured

to a final concentration

with K+-specific

of 4%.

supplemented

et al. (1986). LS medium, containing was performed

to confer K + uptake in S. cerevisiae cells. trklA[YCpSO], trklA[c-TM11

the point of glucose

for plasmids),

with

R1260, R1262 and R1745, respectively,

electrodes

virtually

by the cation method

of Ito

and trklA[u-TRKI] strains R1260, as described

previously

(Gaber

42 TABLE

I

Strains

used

Strain

Source

Genotype

(reference)

E. coli K-12 hsdS20 recA 13 ara- 14

HBlOl

proA

Maniatis

et al. (1982)

IacYl galK2

rpsL 20 xyl- 15 mtl- 14 supE44 A(lac-pro) supE hsdD5 [F’ traD36 proA + B + lacIq

TGI

Amersham

Corp.

lacZAMlS] Yeast

-3

-2

2

3

6

of trkZA[YCpSO], trklA[c-TRKI]

and

0

1

1/[RbCIl G. Fink

R1668

Fig. 4. Kinetics

4

5

-I

s. UYarWvl

(mM-’

)

R757

MATa ura3-52 his4-15 lys9

Gaber

et al. (1988)

cells (R1260, R1262 and R1745, respectively; uptake. shRb + flux assays were performed

R1155

MATa trkldl ura3-52

Gaber

et al. (1988)

(1990) with the following modifications:

S. cerevisiae

hisl- I5 lys9 RI262

MATa trkldl ura3-52

R1260

MATa trkldl ura3-52

grown to logarithmic Gaber

et al. (1988)

Gaber

et al. (1988)

his415 lys9[pRG295-l]

MATa trkZA1 ura3-52

This study

This fragment contains a single, large capable of encoding a protein of 1241 aa Fig. 5). By comparison, the c-TRKl ORF 3705 bp, encoding 1235 aa (Gaber et al.,

241 361

for 10 h in LS starvation

cells/ml containing

his4-15 lys9[pLB8-51

1 121

washed

cultures of S. cerevisiae cells were twice in double-distilled

medium.

glucose was added (final concentration

his415 5vs9[YCpSO] R1745

starved

phase,

under

constant

agitation

H,O

and

At the start of each assay,

4%) to a suspension

of 4.0 x IO’

in 50 mM Tris’succinate

pH

5.9,

RbCl and 0.1 pCi/ml ofX6RbC1. RbCl uptake oftrklA[c-TRKI]

and frklA[u-TXKI]

cells was measured

1.0-25 mM RbCl was used to measure

ORF of 3723 bp (approx. 136 kDa, is slightly smaller, 1988). The overall

trklA[u-TRKI]

Table I) measured by RbCl as described by Ko et al.

using

0.2-5.0mM

RbCl and

of trklA[YCpSO] cells.

uptake

nt sequence identity between the u-TRKI and c-TRKl ORFs is 77p/, . In regions of aa identity (described in section e, below), the nt sequence identity is 86%) indicating divergence of nt sequences between conserved regions.

T*TTTTCCCTAATTTATCGCAGTCCATTATT*TTTCT*TACG-GAGAGGAGGGTATCCGTTGGCTCTC~GGAGGAGGTCATTCCTACCC*TTTTTAGCCTGATTTGTT~TTT*CCG*CG AAAAAATCCTTCGGCCACAAATTTCGTGATTTCATTGCTCTATGTGGCCAC TAGTCAGCAATGCACATTAGAGGGACCATGAGCAGAGTGCCCACATTAGCATCGTTCGAAGTACGATAC FEVRYKKS F G H K F R D F RVPTLAS IALCGH R G TM S M H I TATATTTTCCCTAATTTTATCGCAGTCCATTATTTCTATACGATAGTCCTCACGCTAATRACCTCTATTCTGTTGTATCCAGTTAAGAACATCAGATAT TATTGCTCCCCAATTAAAAAA IAVHYFYTIVLTLITS ILLYPVKNIRY I F P N F K K Y Y c s P I ATCGACGCTTTGTTTTTGGCAGCAGGTGCGGTCACTCAAGGTGGTTTGAATACTGTGGATGTCAACAATCTAACCTTATATCAACAGATTATTTTGTACATTATATGCTGCATATCAACG

37 71

IDALFLAAGAVTQGGLNTVD CCCATTGCCGTTCATAGTTGTTTGGCATTCGTAAGACTCTATTGGTTTGAACGTTATTTTGACGGTATTAGAGACTCGTCCAGGCTAAATTTCAAAATGAGAAGAC-GACAATCTTG

111

481

157

601

D G I R D SSRLNFKMRRTKTIL IAVHSCLAFVRLYWFERYF P G-GGGAACTAACCGCTAGAACCATGACCAARAGTAAGACAGGAGGGACTCAAAGAGTGTCACGTCCCGGGAAATCAGACAAGAGAGACGATTTCCAAGAAAAATTGTTTAATGGAGAA KTGGTQRVS RPGKSDKRDDFQEKLFNGE ERELTARTMTKS ATGGTTAATAGAGATGAGCAGGACTCGGTTCATAGTAGCCACAATTCTCGTGATAGTAATAGCAATGCCAATACCAATACC~TAGTAGC~C~T~TAGCATCAACCACAATGGTAGTAGTGGC SHNSRDSNSNANTNS SNNNSINHNGSSG S" H s MVNRDEQD

197

721 841

AGTTTGGATGATTATGTTAGGGAAGACAAAGCGGATGAGGTCGAAAAATACCACGGAAATAAGTCCTATTCAAGTGTAGGTAGTTCGTCCARTACAGCCACAGATGAAAATATAAGTCAA

961

S V G S S KYHGNKSYS SNTATDENISQ SLDDYVREDKADEVE AAATTAAAGCCAAGTAGTCTTCGATTCGATGAGTCGCAAAATAAAAGAAAACGTACGGGAGCCCCTTCAGRAAAATTTGCCAAAAGGAGAGGTTCAAGAGATATCACTCCTGAAGATATG SQNKRK RTGAPSEKFAKRRGSRD SLRFDE K L K P S

VNNLTLYQQI

ILYIICCIST

1081

TATCGATCCATTATGATGCTACAAGGTGAGCACGAGCACGAGGGTACTGCAG~GATG~GGTCCACCTTTAGTTATTGGATCGCCTACGGATGGTAC~G-TATGGAC~CGGTAGTGAGTCA TAEDEGPPLVIGSPTDGTRNMDNGSES YRSIMMLQGEHEG

1201

AAGTCTGCTCCCACGATGAATGAGAGTAAAATCAGGATTCAGGATAAAGGAGCTAAAATTAGCCTTGACCAGGACTCCGTATTACATAGTTCAAATTCTTCCGCATGCACTTCGGATGAG

1321

ISLDQDSVLHSSNSSACTSDE KSAPTMNESKIRIQDKGAK GATTCTCTACCTACAAATTTTGGAGGCACAACACCTTCACCTTCATTGAGCGC~GCCACGGG~TC~GTTCAGGCCC~TAGCGTTCACTG~GGGG-TGCTGATAG-C~GGCCCA SLSAKPRESSSGP IAFTEGENADRKQGP DSLPTNFGGTTP

1441

TCGATTCAGTTCAATATCACTACCGCCAAGGAAGGCTTCTAAATCGAAGAGGGTTTCTACTATGGATGACTTGAACCCAAGATCAATTTTTCCGCATC TKPPRKASKS KRVSTMDDLNPRSIFPHQKKS S I Q F N I

1561

TACATAATGAAACATTTGCCGAAAGCCCCGTCGGCCAGTAGCAGTGACGCTCTCGATAGAGGGCTCATTTCT

1681

KRRLSTGS IDKNSS SDALDRGLIS YIMKHLPKARRI R Q Q I GGTCTAAATGATGATGATGATGGCAACGAAGGTGATAACATGGAAGAGTACTTTGCCGACRACGAAAGTGGGGATGAAGATGACCGAATGCAGCAATCTGAACCACAGTCTGGATCAGAA FADNESGDEDDRMQQSEPQSGSE GLNDDDDGNEGDNMEEY

1801 1921 2041 2161

(Fig. 5)

237 217 317

ITPEDM

357 397 437

WTCATCGAAAGGA S

517 557

CTTAAATTGAAACAACAGCACAGCACCAATTGCAGCAAAACCTGCATCGTATGTACAAGACGAAATCTTTTGACGATAATCGTTCAAAACCTGTCCTCATGGAAAGGTCTAAGACTATC NLHRMYKTKSFDDNRS KPVLMERS LKLKQQQQHQLQQ GATATGGCTGAAGCTAGAGATTTAAATGAACTTGCCAGGACTCCTGACTTTC AAAAAATGGTTTATAAGAATTGGRAAGCCCCATAGAAAGGGGCTGG DMAEARDLNELARTPDFQKMVYKNWKAHHRNKPNFKRRGW AATGGCAAGATGTTTGAACATGGTCCTTATACATCTGACAGCGACCATAATTATCAGGATAATGGAAATAATAGTAGTATAGTTCACTACGCAGAATCTATTCTACATCGTGATAAT NGKMFEHGPYTSDSDHNYQDNGNNSNS IVHYAESI TCTCATAGAAATGGAAGCGAGATGTTTCTTCAGATTCT~TGAGACCACCTATCCTTTG~TGG-C~TGATCACAGCC-CGATGCT~TGGCTATCCCACCTAC~CGATG~ NETTYPLNGNNDH s H SEDVSSDS RN G SQNDANGYPTYNDE

477

KG

K

T

I

597 637

L

H

R

D

N

677 717

43 2281 2401 2521 2641 2761 2,381 3001 3121 3241 3361 3481 3601 3721 3841 3961 4081

GAGGAAGGCTATTACGGTTTACATTTCGATTCTGATTATAACTTGGATCCTCACCATGCTCTATCCAGTAACGCTAGCAAAAACTACTTGTCATGGCARCCAACCATTGGACGTAATTCA H H AL S S N A S KNYLSWQPTIGRNS EEGYYGLHFDSDYNLDP 757 RACTTTCTTGGGTTGACAAGAGCCCAAAAGGACGAATTGGGTGGTGTCGAGTACAGAGCCATCAAACTACTATGTACCATATTGGTTGTCTATTATGTTGGATGGCACATCGTCTCTTTT ELGGVEYRAIKLLCTILVVYYVGWHIVSf NFLGLTRAQKD 791 GTTATGTTGGTGCCTTGGATTAACTTGAARAAGCACTATAGCGATATTGTCAGAAGCGATGGCGTTTCACCTACATGGTGGGGTTTTTGGACAGCAATGAGTGCCTTCAACGATTTGGGA IVRSDGVSPTWWGFWTAMSAFNDLG VMLVPWINLKKHYSD 837 TTARCATTAACCCCGGACTCAATGATGTCATTTGATAGCTGTATATCCCTTGATCGTTATGATTTGGTTTATCATTATTGGAAATACAGGGTTTCCCATCCTTCTTAGATGCATTATT LTLTPDSMMSFDKAVYPLIVMIWFII IGNTGFP ILLRCII 877 TGGATAATGTTCARACTTTCCCCTGATTTGTCACAGATGAGAGAGAGTTTAGGCTTTCTTTTAGATCACCCGCGTCGTTGTTTTACTCTATTATTTCCAAAGGCTGCCACATGGTGGCTG SLGFLLDHPRRCFTLLFPKAATWWL WIMFKLSPDLSQMRE 917 CTTTTGACGCTTGTGGGACTAAATTTTACAGATTGGATCTTATTTATTATTTTGGATTTTGGATCAACTGTAGTTAAATCGTTATCAAAAGGTTATAGAGTTCTTGTTGGTTTATTCCAA LLTLVGLNFTDWILFII LDFGSTVVKSLSKGYRVLVGLFQ 957 TCTGTCAGTACGAGGACCGCCGGTTTCAGTGTCGTTGATTTGAGTCAGTTACACCCCTCTATCCAAGTCTCCTATATGCTTATGATGTATGTCTCCGTGTTGCCATTAGCTATTTCTATT SVSTRTAGFSVVDLSQLHPSIQ VSYMLMMYVSVLPLAISI 997 AGACGGACAAATGTTTACGAGGAGCAATCTTTGGGACTGTATGGAGAAATGGGAGGTAAACCAGAAGATACTGATACTGAAGAGGACGGCGACTGCGATGATGAAGATGACGACAACGAA SLGLYGEMGGKPEDTDTEEDGDCDDEDDDNE RRTNVYEEQ 1037 GAAGAGGAGAGCCATGAAGGTGGAAGTAGCCAAAGGGGCAAATCAAAG-GAAAC AAAAAAGAAAAAAUGAGAAAAGAAAATGAGAACCCCAACGAAGAATCAACCAAGTCCTTCATT TKKKKKRKENENPNEESTKSFI EEESHEGGSSQRGKSKKE 1077 GGTGCGCATTTAAGAAGACAGCTATCTTTTGATTTATGGTTTTTGTTTCTTGGGCTATTTATCATTTGTATCTGCG~CGTGAC~GAT~GACATCCAGAGACCG~CTTC~TGTA FDLWFLFLGLF IICICERDKIKDIQ GAHLRRQLS RPNFNV 1117 TTTACAATTCTTTTTGAGATTGTAAGCGCTTATGGTACAGTCGGACTATCATTGGGCTATCCGAACACTAACCAATCATTTTCAAGACAGTTAACCACATTATCTAAATTAATCATCATCATA F T I L F E IVSAYGTVGLSLGYPNTNQSF SRQLTTLSKLIII 1157 GCTATGCTGATTAGAGGTAAGAATAGAGGTCTGCCCTATTCTTTGGACCGTGCTATTATCCTGCCAAGTGATAGACTTGAACATATTGATCACATTGAGGACTTGAAATTGAAGAGACAG ILPSDRLEHIDH AMLIRGKNRGLPYSLDRAI IEDLKLKRQ 1197 GCCAGAACCGATACGGATGATCCGATGACGGAGCATCTCRAGAGAAGCATTAGCGATGCCRAACATCGTTGGGACGAACTTAAACACAAGAGAAGCCTTTCCCGGAGTTCGAAAAGAAGC SDAKHRWDELKHKRSLSRSSKRS ARTDTDDPMTEHLKRSI 1237 ACCAAAACCAACTARACTACTTCGCCCATACCACGATACTAATACATATGGAAATGTATCTATCAAAAGTTATTGCATGTATTTTTTTTCTAGGATTATACATAGACTTCTTAACTA T K T N * 1241 TTAGAATATCTTCTGTACTTACCTTCGTGCATTCTTTCTCGATGTTTTTTTTTCCTGCGATGCCTTCACTTTTCTTTTTTTGGGTCCCTT~GACGGCCAGATCGCTTGRAACTAATGT ACAATATACAATGTCTAGA 4099

Fig.5. Nucleotide Sequence of the S. UYUII(Mu-TRKZ gene. The nt are numbered

on the left and aa are numbered

on the right. A TATA sequence

located

have been submitted

and assigned

at nt 34-37.

M57508.

Sequencing

generated

by Sau3A

facilitate

sequence

An asterisk

indicates

the 5-kb fragment

the stop codon. The nt and aa sequences containing

u-TRKZ was performed

and TaqI digests of the u-TRKZ clone, were inserted analysis,

exonuclease

III was used to generate

nested

(e) Comparison of Saccharomyces uvarum and Saccharomyces cerevisiae TRKl aa sequences A comparison of the deduced aa sequences of u-TRKl and c-TRKl is presented in Fig. 7. For optimal alignment, live insertions and three deletions were included in u-TRK 1. The overall identity between the two proteins is 78 % ; they show 86% similarity when conservative substitutions are included. Examination of hydrophilicity plots (Hopp and Woods, 198 I), generated from the c-TRKl and u-TRKl aa sequences, revealed a nearly identical pattern of alternating hydrophobic domains separated by hydrophilic regions (Fig. 6). These results are consistent with the presence of twelve membrane-spanning domains in each of the K’ transporters according to the criteria of both Eisenberg (1984) and Rao and Argos (1986). Within the putative transmembrane domains, the sequence identity between c-TRK 1 and u-TRK 1 is 95 % . In contrast, sequence conservation between the non-transmembrane regions of these proteins shows only 74% identity. Four of the transmembrane regions are 100% identical and the few substitutions in the remaining eight are mostly conservative. Given the ability of u-TRKl to confer high-affinity uptake in S. cerevisiue, the relatively strict conservation of aa comprising the putative membrane-spanning domains suggests that these regions play an important role in high-affinity K’ transport. Significantly less conservation is observed between the transmembrane domains of

by the chain-termination into the pGEM4.Z sets of deletions

method

of Sanger

vector (Promega,

Madison,

of some fragments

Fig. 6. Comparison Hydrophilicity method

to GenBank

of the hydrophilicity

plots of the deduced

of Hopp and Woods

hydrophobic

(Henikoff,

character

the accession

et al. (1977). DNA fragments, WI) for sequence

analysis.

To

1984)

plots for c-TRKI

aa sequences

and u-TRKl.

were generated

(1981). Two algorithms,

(Eisenberg,

is

number

by the

one based on the

1984) and one based on membrane-

buried helix parameters ofaa (Rao and Argos, 1986) were used to identify putative transmembrane domains of the S. cerevisiae and S. uvarum K + transporters.

44

the high- and low-affinity K’ transporters of S. cerevisiue (75% identity) (C.H. Ko and R.F.G., manuscript in preparation), further suggesting that these regions confer high

found

affinity for K + . Cysteines located near a membrane-spanning

residues in c-TRK 1 and TRK2, the low-affinity K + transporter in S. cerevisiae, and each is located in or near a transmembrane domain (C.H. Ko and R.F.G., manuscript

domain

in

are completely

in preparation). In the case of the nicotinic

(Numa et al., 1987). The deduced aa sequence of c-TRKI contains nine Cys residues, each of which is located in, or adjacent to, putative transmembrane regions. Since the Cys

u-TRKl

positions

conserved

in

u-TRKl, it is likely that these residues are important for TRKl function. In addition, there are four conserved Cys

the a-subunit of the Torpedo californica acetylcholine receptor are required for proper conformation of the receptor

c-TRKl

at these

T. californica, negatively

transmembrane

domain

acetylcholine

receptor

in

charged aa residues adjacent to a are major determinants of the rate

100

MHFRRTMSRVPTLASLEIRYKKSFGHKFRDFIALCGHYF~~YIFPSFIA~~~ISLTLITSI~YPIK~RYIDT~~GA~~LN~~~ II:I IIIIIIIIII:l:IIIIIIIlI/l llI/IlIII ./:IIlIII.IIIIIIllll III/IIIIIII:II.IIIl.IIIIIIIIIlIllllll:II 1 MHIRGTMSRVPTLASFEVRYKKSFGHKFRUFI~CGHYCSPIKKYIFPNFIAVHYFYTIVLTLITSILLYPVKNIRY~~GA~O~L~~ 1

100

. 101

LSLYQQIVLYIVCCISTPIAVHSC

LAFVRLYWFERYFDGIRDSS

IIIIIIIIIIIIIIIIIIIIII.:I

IIII.

I I .I

Illlllll~llIIl

101

LTLYQQIILYIICCISTPIAV?ISCLAFVF!IXWFERYFDGIRDSSRLNFKMRR

200

RDEQDSVHSDQNSHDISRD.....

SSNNNTNHNGSSGSLDDFDETDDNGEYQENNSYS~GSSS~~ESLNQ~~SSLRFDEPHSKQRPARVPS

IIIIIIlII.:II:I

I.lI..IlIIIIIIIII:I:II..I:

..:

199

RRNFKMRRTKTILERELTARTMTKNRT.GTQRTSYPRKQAKTDDFQEKLFSGEMVN

I.IIIII:III:IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII

200

TKTILERELTARTMTKSKTGGTQRVSRPGKSDW(DDFQEKLFNGEMVN

:.I::I.IIl.IIIIIlI..II.:.Il

294

IIIIIIIII.:.I.:..

.II

201 RDEQDSVHSSHNSRDSNSNANTNSSNNNSINHNGSSGSLDDYVREDKKYHGM(SYSSVGSSSNTADENISQKLKPSSLRFDESQNKRKRTGAPs

300

295 EKFAKRRGSRDISPADMYRSIMMLQGKHEATAEDEGPPLVIGSPADGTRYKSNVNKW(ATGINGNKIKIRDKGNESNTDQNSVSSEANSTAsVSDEsSL

394

IIIIIIIIIIII.I.IIIIIIIIIII.II:IIIIIIIIIIIIII.IIII

..I...

I.I..:I:.II:I.III..

..II.I:.III.II

. Il:II

301 EKFAKRRGSRDITPEDMYRSI~EHEGTAEDEGPPLVIGSPTDGTRNMDNGSESKSAPTSKIRIQDKGAKISLDQDSVLHSSNSSACTSDEDSL

400

395 HTNFGNKVPSLRTNTHRSNSGPIAITDNAETDKKHGPSIQFDITKPPRKI..SKRVSTFDDLNPKSSVLYRKKASKKYLMKHFPKARRIRQQTG

492

.IIII...III....:

. . . II.11

IIIIIl:IIIII:I

I.IIIII:I:..:.I:I:IIIIII:IIIIIIl

I:IIl:IIIIlIIIIlIIIIIII 500

401 PTNFGGTTPSLSAKPRESSSGPIAFTEGENADRKQGPSIQFNITKPPRKASKSKRVSTMDDLNRSIFPHQKKSSKGYIMKHLPKARRIRQQIKRRLSTG LHRMYKTKSFDDMlSRAVPMERSRT

493 SIEKNSSNNVSDRKPITDMDDDDDDDDNDGDNNEEYFADNESGDEDERVQQSEPHSDSE~SH~~QL~N Il:IIIl.:. I/ I.:: :IIII:I:III IIIIIIIIIIIII:I:IIIII:I:IIII .IIII 501 SIDKNSSSDALDRGLISGL..NDDDDGNEGDNMEEYFADNSGDEDDRMQQSEPQSGSEL..HQLQQN

IIIIIIIIIIIIIIIIIIIII::I

LHFMYKTKSFDDNRSKPVLMERSKT

NFRKRGWNNKIFEHGPYASDSDRNYPDNSNTGNSILHYAESILHHDGSHKNGSEEASSDSNENIYS

593 IDMAEAKDLNELARTPDFQKMVYQ-

592

IIIl:I 596 692

IIIIII:IIIIIIIIIIIIIIII.IIIIIII.IIII::IIII.I:IIIIII.IIII:II.II:I.:III:llllllll:l.ll:llll:.llllll..l. 597 IDMXARDLNELARTPDFQKMVYKB

NFKRRGWNGKMFEHGPYTSDSDHNYQDNGNNSNSIVHYAESILHRDNSHRNGSEDVSSDSNE~YP

790

693 TNGGSDH..NGLNNYPTYNDDEEGYYGLHFDTDYDLDPRHDLSKgsgktY~WQPTIGRNSNFLGLTRAQKDELGGVEYRAIKLLCTILVVYnrGWHIVA

II..II

I:

696

I.IIIIIIIIIIIIIIIIIIIIIlllllIIIIIIIIIIIIIIIIIIIIIIII.

I.IIlIII:IIIIIIIIII.II:III:I.II...:

697 LNGNNDHSQNDANGYPTYNDEEEGYYGLHFDSDYNLDPHWIVS

796

791

890

797

896 YMLMMYVSVLPLAIS

891 LLDHPRRCFT~PKLLT~G~ITDWILFIILDFGSTVVKSLSKGYRVLVGLFQSVSTRTAGFSVVDLSQL~SIQVS

990

lllllllllllllllIIIIIIIIII.III:llllllllllllllllllIIIlIIIIIIIIIIIIIIIIIlllllllll.lllllllllllllllllllll 897 ~DHPRRCFTLLFPKAATLLTLVGL~TDWILFIILDFGSTVVKSLSKGYRVLVGLFQSVSTRTAGFSWDLSQL 991 IRRTNVYEEQSLGLYGDMGGEPEDTDTED~G..

lIIIIIIIIIIIIIII:lII.IIIIIII:II

996

.NDEDDDEENESHEGQSSQRSSS NNNNNNNRKKKKKKKTENPNEISTKSFIGAHLRKQLSFDLWFLF

:::II:II:IIIII.IIlI:.I

..:.:

IIII:I..IIIII

997 ;CRRTNVYEEQ~LGLYGEMGGKPEDTDTEEDGDCDDEDDEDDD~~ESHEGGSSQRGKS...KKETKKKKKRKENENPNEESTKSFIGAH~

1093

1088 LG~IICICEGDKIKDVQEPNFNIFAILFEIVSAYGTVGLSLGYPDTNQSFSRQFTTLSKLVIIAMLIRGKNRGLPYSLDRAIILPSDRLEHIDHLEGMK

IIIIIIIIII

IIIIl:I

1087

IlIIIIIIIII:IIIIIIIIII

1187

IIII:I.IIIIIIIIIIIIIIIIIII:IIlIIIII:IIlIII:IIIIIIIIIIIIIIIIIIIIIIIIIIlllllll:l::l

1094 I&hFIICICERDKIKDIQRPNFNVFTILFEIVSAYG~GLSLG~NTNQSFSRQLTTLS~IIIAMLIRGKNRGLPYSLDRAIILPSDRLEHIDHIEDLK 1188 LKRQARTNTEDPMTEHFKRSFTDVKHRWGALKRKTTHSRNPKRSSTTL

IIIIIII:I:IIIIII:III:.I.lIII:.II:I

1235

. II..III..I

1194 LKRQARTDTDDPMTEHLKRSISDAKHRWDELKHKRSLSRSSKRSTKTN Fig. 7. Comparison of deduced aa sequences

1193

of u-TRKl

and c-TRKI.

1241

Vertical lines indicate

identity,

two dots indicate

a conservative

substitution

and

single dots indicate semi-conservative substitutions. Lower-case letters indicate the putative nt-binding

Potential membrane-spanning domains are underlined once and charged regions are underlined twice. domains. The PEST containing regions in c-TRKl are aa 323-342, 507-539, and 994-1032 with

PEST-scores

(Rogers

of 5.66, 21.79 and 18.97, respectively

et al., 1986).

45 of ion transport (Imoto et al., 1988). This may also be true for other Iigand-gated channels such as the neuronal acetylcholine receptor (Boulter et al., 1986), the GABA receptor (Schofield et al., 1987) and the Gly receptor (Grenningloh et al., 1987). Clusters of charged aa in c-TRKl and u-TRKI could have an anaiogous function in determining K’ affinity or rate of transport. A comparison of the u-TRKl and c-TRKl aa sequences revealed that four highly charged regions are conserved: two positive and two n~gati~~e domains (Fig. 7). There is a positive and a negative region in each of two large hydrophilic domains between M3 and M4 and between M 10 and Ml 1. Charged regions within membrane proteins may mediate interactions that are important for proper folding or may function directly in protein activity. Hartmann et al. (1989) have proposed a method to predict the orientation of the first transmembrane domain of integral membrane proteins. This scheme predicts the N termini of c-TRKl and u-TRKl to be cytoplasmic. If the model predicting twelve transmembrane domains is correct, the large hydrophilic region of both transporters would be extracellular. The putative extracellular location of this domain is inconsistent with the observation that, in c-TRKI, this region contains a potential nt-binding domain (Gaber et al., 1988). The analogous region in u-TRK 1, however, is completely dissimilar (Fig. 7). Thus, the simil~ity between the potential nt-binding sequence in c-TRKI and the consensus sequence found in authentic nt-binding proteins (Higgins et al., 1986) may be fortuitous. Although it is possible that this region of c-TRKl is part of an authentic nt-binding domain, the ability of I(-TRKI to confer highaffinity uptake in S. cerevisiae suggests that nt binding is not essential for K + transport. Both u-TRKl and c-TRKl contain sequences that are strongly predicted to target proteins for rapid degradation. Rogers et al. (1986) have observed that some proteins with short intracellular half-lives contain one or more regions rich in Pro (P), Glu (E), Ser (S) and Thr (T) that are flanked by positively charged aa (Rogers et al,, 1986). An algorithm that ranks an aa sequence according to its PEST composition, was applied to c-TRKI. Using the criteria that a PEST-score greater than or equal to five may provide a signal for rapid degradation, three PEST sequences with scores of 5.66, 18.97 and 2 1.79 were found in c-TRKl (Fig. 7). All of these regions are highly conserved between c-TRK 1 and u-TRK 1. In addition, proteins containing ArgArg pairs are also rapidly degraded (Rogers et al., 1986). c-TRKl contains six Arg-Arg pairs that are completely conserved in u-TRKl (Fig. 7). These signals for rapid degradation in c-TRK 1 and u-TRK 1 may regulate the amount of protein present in the cell (Rogers et al., 1986). Since integral membrane proteins, like TRKl, contain many hydrophobic domains, if they do not reach the plasma

membrane within a short time after synthesis it may be necessary to degrade them to prevent interfereIice with cytosolic activity. Another possibility is that K * transport is regulated by this degradation signal. Since the degradation of PEST-containing proteins is thought to be mediated by the Ca2’ -activated protease calpain (Rogers et al., 1986), ionic signalling through Ca2+ might alter the K + concentration in the cell. When Ca2 + levels increase, calpain is activated and the K’ transporter is degraded, thus reducing the amount of K + being transported into the cell. (f) Conclusions

(1) Most Saccharomyces species contain a high-affinity K ’ transporter gene highly related to TRKl in S. cerevisiz The TRKI homologue found in S. ~va~rn appears to have undergone the greatest evolutionary divergence. (2) u-TRKl suppresses the K’ transport defect of S. cerevjs~~etrkld cells by conferring high-a~nity uptake of this ion. (3) u-TRKZ encodes a membrane protein of 1241 aa that is 78% identical and 86% similar to c-TRKl. (4) The relatively strict conservation of aa in the transmembrane domains suggests that these regions play an important role in high-affmity K’ transport.

ACKNOWLEDGEMENTS

We wish to thank D. Stillman for providing us with info~ation on the PEST sequences and M. Vidal and C. Ko for helpful discussions. This work was supported by Grants from the National Science Foundation (DCB8711346 and DCB-8657150) to R.F.G.

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