Heterologous Expression of Genes in Bacterial ...

Annu. Rev. Plant. Physiol. Plant. Mol. Biol. 1995.46:419-444. Downloaded from www.annualreviews.org Access provided by WIB6013 - Freie Universitaet Berlin - FU Berlin on 09/21/15. For personal use only.

Annu. Rev. Plant Physiol. Plant Mol. B ioi. 1995.46:419-44 Copyright © 1995 by Annual Reviews Inc. All rights reserved

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HETEROLOGOUS EXPRESSION OF GENES IN BACTERIAL, FUNGAL, ANIMAL, AND PLANT CELLS Wolf B. Frommer and OlafNinnemann Institut flir Genbiologische Forschung,

D-14195 Berlin, Gennany

KEY WORDS: e xpression cloning, functional expression, complementation, yeast, Xenopus oo cyte

CONTENTS ABSTRACT..................................... ........ . . .. ................................ ............................................

420

INTRODUCTION .................................... .. ....... ........................................................................

420

How Similar Are Organisms? ................. .. .. . . ... . . . . . . . . ... .... ..... ... ....... . .... ... . .... . . . . . ... ... ... . ... . .....

Ways to Isolate Genes 0/ Interest..... .... . . . . . . . . .. . . ........ . . . . . . . . . . . . . . . . . . . . .... .... . . . . . . . . .... ....................

421 422

EX PRESSION OF FOREIGN GENES IN BACTERIA...........................................................

422

EX PRESSION OF F OREIGN GENES IN FUNGI...................................................... ............

423

History o/ Yeast Complementation .. . ... ... ... .. ................ ............ .... ...... ..... .. .. .. . . . . . . . . . . . ........ . . .. How to Isolate a Gene by Complementation .... ... .... .... ... . .. . . . . . . . . . . . . .. .. .. .. ..... . ......... ... . ... . .. . . .

423 423

Isolation 0/ Enzyme-Encoding Plant Genes by Complementation . . . . . . . . .. ... . . .... ........ ......... . . Functional Expression 0/Membrane Proteins in Yeast .. ...... .... ... . . .. . . . . . .. .... ... .. .......... ... . .... .

424 424

Isolation o/Transporter Genes ............. ..... . . . . . . . . .. .......... ........... . . ... ... . . .. . . . ................ ....... .....

428 429

Extending the Scope o/ Complementation. ... . . . . . . . .................... ....... . .. ...... ........ . . . . . . . . . ... ... . .... Applications/or Structure-Function Studies .. ... . . . . . . . . . . . . . . ............ ........ ..... . ........... . . . . . . . . . .. .... Heterologous Promoters in Yeast . . . . ........ .... ....... .. ...... . . . . . ........ . . . . . . .. .. .. ...... ..... . ....... .... .. . ......

429 430

Requirements/or Heterologous Complementation 0/ Mutations in Yeast . .... ......... ...........

430

EX PRESSION OF FOREIGN GENES IN ANIMAL CELLS................... . . ...... ....................

432

Transient Expression in Xenopus laevis Oocytes ........... ..... .... ............... .. . . . . . . . . . . . ........ . ... .. ..

432 433 Expression in Insect Cell Cultures .. . ....... . ................................ .... ........ ........ .... .................... 435 Expression in Mammalian Cells .. .... ...... ........ ... .. ........ ........ ... . .. . . . ... . ...... . .. . . .. . . . . . . . . . . . . . . . . .. . . . . . 435 Characterization 0/ Plant Transporters in Oocytes..... . . . . . . . . ................... . . . . . . . . . . . . . . . . . . . . . . . . . . . ..

EX PRESSION OF GENES IN PLANT CELLS. .. . ...................... .................... ........ . . ...............

436

CONCLUSIONS........................................................................................................................

436

0066-4294/95/0601-0419$05.00

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ABSTRACT

Analysis of gene function is of central importance for the understanding of physiological processes. Expression of genes in heterologous organisms has allowed the isolation of many important genes (e.g. for nutrient uptake and transport) and has contributed a lot to the functional analysis of the gene products. For animal research, expression in Xenopus oocytes and cell cultures are predominant techniques, whereas in the plant domain, yeast has become the prevalent expression system. This review provides a survey of this quickly developing field and intends to assist researchers in determining appropriate experimental approaches for specific biological questions. Because heterolo gous expression technology is of special value for the analysis of proteins that are difficult to handle biochemically, the examples given concentrate on mem brane proteins, i.e. transporter proteins. Also included is a detailed discussion of the functional expression methodology and its use in identifying and char acterizing genes and proteins.

INTRODUCTION In recent years plant biology has made rapid progress through the use of joint approaches from different disciplines such as physiology, biochemistry, genet ics, and molecular biology. Significant progress has been made in the under standing of fundamental processes such as nutrition, metabolism, and trans port. Whole plant studies are extremely powerful and have enabled a better understanding of the capacities of plants. However, these studies give only a crude picture of the underlying mechanisms because plants are so complex. Biochemical studies are often limited as well. Kinetic analysis often uncovers only the activity of the major stable components; the presence of multiple systems often makes it difficult to resolve complex kinetics. Thus, the analysis of single, isolated systems is desirable. In many cases, especially in transport physiology, methodology is limited. Genetics has always been instrumental for studying plant physiology, although the wealth of techniques available in bacterial or fungal genetics has not been routinely used because of the greater complexity of plants and because of technical limitations. The existence of plant mutants with distinct phenotypes does not always enable researchers to determine causes of the observed defects. Such determinations require new methods for isolating and characterizing individual proteins. Furthermore, the need for techniques to determine gene-product function has become more important because many genes with unknown functions are being identified through random and genome sequencing projects. Heterologous expression of plant genes provides a new technique for deter mining gene-product function. This technique, which has been used success fully for the analysis of many mammalian genes, will be especially valuable

HETEROLOGOUS EXPRESSION

421

for the analysis of plant functions for which no mutants are available and for which no screening scheme or phenotype is predictable. For example, until recently, most researchers thought that membrane permeable substances, such as water and ammonium, did not require transport proteins. Carriers for these substances were identified by heterologous expression cloning assays (115,


128). This review covers expression systems for both animal and plant genes. Emphasis is on functional expression and expression cloning techniques using yeast, Xenopus oocytes, and other animal cells. Because this approach has been most powerful in identifying genes that are otherwise difficult to define, such as integral membrane proteins, identification of transporters is covered in detail.

Ways to Isolate Genes ofInterest In the simplest case, PCR or heterologous screening techniques allow isolation of known genes from the organism of interest. The growing pool of informa tion from random cDNA sequencing projects increases the probability of identifying genes by computer-based searches. Because it is not always possi ble to deduce gene function from the sequence, methods are still required to identify the role of the encoded proteins. Even highly related genes may have different functions as is the case with genes for plant nitrate-, amino acid-, and peptide transporters (59, 174, 182) or mammalian Na+ -coupled transport sys tems (134). If no related gene sequence is available, the classical biochemical approach for isolating the gene is to develop an in vitro assay for protein activity that enables purification. The purified protein can be used for immu noscreening of expression libraries or for N-terminal sequencing and sub sequent isolation of the gene using oligonucleotides. The purification of some proteins can be problematic because of instability, low abundance, lack of appropriate assays, or difficulties in purification. Many of these problems occur during the purification of membrane proteins. The only biochemical detection system for transporters are transport assays, which demand the pres ence of two compartments. Functional reconstitution is therefore necessary, and this difficult technique has been successful in only a few cases. These difficulties may explain why little is known about transport proteins, espe cially in multicellular organisms. Another tool for the isolation of transporters is the use of radiolabeled ligands or inhibitors that interact with the desired protein and hence facilitate protein purification. This technique allowed identification of the first transport protein from plants, the triose phosphate translocator (50). Methods that cir cumvent problems associated with biochemical identification procedures in clude, for example, isolation of membrane proteins by two-dimensional elec trophoresis (56) from tissues that differ in their transport capacity or by immu nological approaches with antisera directed against membrane vesicles

(64).

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Specific antibodies can be identified by their ability to inhibit transport proc esses and subsequently can be used to isolate the proteins from expression libraries (90). Reverse genetics (i.e. the identification of genes starting from specific mutants) represents an alternative (163). Unfortunately, mutants or screening Annu. Rev. Plant. Physiol. Plant. Mol. Biol. 1995.46:419-444. Downloaded from www.annualreviews.org Access provided by WIB6013 - Freie Universitaet Berlin - FU Berlin on 09/21/15. For personal use only.

protocols are not available for all genes of interest. An efficient way to clone and simultaneously prove the function of a gene is functional expression in heterologous host cells. Complementation of mutants has been performed mainly in unicellular organisms. Essential prerequisites are the existence of suitable mutants and methods for selecting transformants. One advantage of expression cloning is the high probability of identifying full-length clones, thus allowing functional analysis.

How Similar Are Organisms? Heterologous expression systems are based on the assumption that the basic principles of protein expression and function are similar in all organisms. The sequences of most eukaryotic proteins are well conserved (24). Eukaryotic organisms share many principles of cell compartrnentation, intracellular trans port, and regulation, such as vesicular trafficking along the secretory pathway

(13, 17), cell-cycle control (13 1), signal transduction (8, 84), and chromatin structure (60). Nevertheless, important differences exist between fungal, plant, and animal cells in terms of the presence and composition of cell walls, and the presence of specialized organelles such as plastids and vacuoles. Regarding energization of secondary active transport processes at the plasma membrane, plants are more similar to yeast than to animal cells because plants and yeast use proton gradients, whereas animals use mainly sodium gradients. Multicel lular organisms, however, have many properties for which no equivalent exists in unicellular organisms, such as intercellular communication across cell walls and through signals transferred in the vascular system in plants, or electrical and hormonal long-distance communication in animals.

EXPRESSION OF FOREIGN GENES IN BACTERIA Classical molecular genetics has been developed for bacteria (especially Es

cherichia coli), and suitable expression vectors and hosts are available. The advantages of heterologous expression in E. coli include the availability of well-established molecular tools and defined mutants, as well as high growth rates and high yield of overproduced protein. As early as 1976 (177), an E. coli histidine auxotrophy was complemented by yeast DNA. Since then, several eukaryotic genes have been isolated and characterized by heterologous expres sion in bacteria (66, 76). In 1986, the first plant gene was identified by complementation of the ginA E. coli mutant (35). Since then, many other plant


423

genes have been used to complement E. coli mutations (71, 137, 171). Disad vantages of the bacteria are the lack of organelles and cellular modification mechanisms responsible for the types of RNA and protein processing found in eukaryotes. Furthermore, eukaryotic polypeptides expressed in E. coli often denature and aggregate (12), and many membrane proteins, even from E. coli


itself, are toxic when overexpressed in bacteria (60).

EXPRESSION OF FOREIGN GENES IN FUNGI

History of Yeast Complementation In 1978, the development of yeast transformation provided a new way to isolate eukaryotic genes (77). Shortly thereafter, the development of shuttle vectors allowed complementation of the yeast leu2 mutation with E. coli DNA (15). One year later, a leu2/canl mutant was utilized to isolate the yeast arginine permease gene (25). In 1981, the first heterologous complementation with eukaryotic DNA was performed with a genomic library from Drosophila melanogaster in the yeast ade-8 mutant (75). The presence of intervening sequences in genomic DNA from higher eukaryotes turned out to be problem atic (16, 118); therefore, cDNA libraries have become the predominant tool for complementation. The first gene of a higher eukaryote isolated from a cDNA library by complementation was the human homologue of a yeast gene con trolling the cell cycle (06). Since then, functional expression has been used frequently to prove the function of genes or to isolate new genes (49). Functional expression in yeast also provides a source for preparative protein production for pharmaceuticals or for enzymatic synthesis of biochemicals.

How to Isolate a Gene by Complementation Various approaches can be used to identify specific clones by transforming appropriate yeast mutants with plasmid-borne genomic or cDNA libraries (44, 151). The simplest strategy is the complementation of a recessive mutation, which includes the following steps: (a) Obtain or construct the appropriate strain that carries the mutation of interest and contains a selectable marker, normally an auxotrophy. (b) Establish a selection or screening system. (c) Clone the cDNA library into a shuttle vector carrying a gene that complements the auxotrophy (several libraries are already commercially available). (d) Transform the yeast mutant with the cDNA library. Several transformation protocols are available, but protocols that allow storage of competent cells are advantageous [transformation rates of approximately 104/g DNA (37)]. (e)

Either select first for the presence of auxotrophic marker and then replica plate on selective media or directly perform a double selection. if) Replate positive


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FROMMER & NINNEMANN

colonies, isolate plasmid DNA, and retransfonn to exclude artifacts resulting from reversion of the mutation in the yeast strain. If appropriate mutants are not available, specific mutations can be intro duced by gene disruption (152). Synthetic lethals can be used instead of mutants (118, 147, 149). Increased resistance to toxic compounds may provide an alternative selection procedure. The use of mutants is simple but requires a change in the selectable phenotype, such as differences in growth. Screening by functional assays can be an alternative if no selectable phenotype is avail able (33). Numerous reports demonstrate the efficiency of functional expression of already identified genes in heterologous hosts, especially for analyzing mam malian genes involved in a broad range of cellular functions (Table 1). Het erologous expression in yeast has proved useful in the analysis of genes involved in human diseases (e.g. p53) (85), defects causing galactosemia (55), and enzyme testing with pharmacological agents (140). Comparable experi ments using plant genes may provide a screen for potential herbicides based on their ability to interact specifically with a known protein. On the other hand, heterologous complementation has also been used successfully to isolate new genes, including those catalyzing specific metabolic and regulatory steps (Ta ble 1).

Isolation of Enzyme-Encoding Plant Genes by Complementation The first attempt in 1982 to complement yeast auxotrophic markers with a cosmid library from Arabidopsis thaliana was not successful (191). A decade later, two plant cDNAs were functionally expressed in yeast (3, 70) to confirm their assumed in vivo functions. One year later, Minet was able to show that several A. thaliana cDNAs could complement Saccharomyces cerevisiae auxotrophic marker mutations (123). Since then multiple reports have been published on cloning of genes that encode soluble proteins (Table 2).

Functional Expression ofMembrane Proteins in Yeast Since 1986, yeast cells have been used as functional expressions systems for membrane proteins of bacterial and animal origin (Table 1). For plant genes, yeast has become the preferred expression system (Table 2). Heterologous systems can be used if the respective function is lacking in the host as in the case of Na+,K+-ATPases in yeast (81) or active glucose transport in Schizosac charomyces pombe. Glucose transporters were the first plant transport proteins to be functionally expressed in S. pombe (28, 156, 157). The proteins are targeted correctly to the plasma membrane. The A. thaliana H+-ATPase AHA2 partially complemented the S. cerevisiae ATPase pmal mutation. The protein is functional, but a large proportion is trapped in the endoplasmic reticulum (ER). Removal of the C-terminal domain of AHA2led to increased



Table 1

Expression of animal proteins in heterologous hosts

Functional expression in yeast

Reference

Vertebrate transcription factor JUN

176

Human cell cycle p34CDC2 and CDK2 kinases

183

Mammalian type IV phosphodiesterase

140

Chicken calmodulin

\32

Human galactose I phosphate uridyltransferase

55

Rabbit

15

a

globin

Chicken actin ACT!

93

Mouse DNA topoisomerase-TOP2

2

Archaebacterial bacterio-opsin

105

Human multi-drug resistance protein MDRt

102

Mouse P-glycoprotein

154

Human estrogen receptor

120

Human mitochondrial anion channel

21

Mammalian Na+/K+ ATPase

81

Human Cytochrome P450

142

Rat M5 muscarinic acetylcholine receptor

83

Torpedo nicotinic acetylcholine receptor

61, 195

N(phrops 16-kDa ductin

80

Cloning by complementation in yeast Human purine biosynthetic proteins

123,161

(ADEl , 2, 3, 8 homologues) Human dihydroorotate dehydrogenase

122

Human pyrroline 5 carboxylate reductase

38

Human glycogen branching enzyme

181

Rat cAMP phosphodiesterase

31

Human cell cycle control protein CDC2

106

Human DNA ligase I

11

Human CCAA T binding protein

12

Xenopus N-ras

172

Cloning by suppression of phenotype in yeast Human chaperonin-like protein

164

Functional e x pression of membrane proteins in oocytes E. coli glycerol transporter GLPF

116

Mammalian Na+/glucose transporter SGLTI

72

Mammalian Na+/myo-inositol transporter

104

Mammalian Na+/nucleoside transporter

134

Mammalian glucose transporter GLUTl -7

167

Rabbit urea transporter UT2

196

Rabbit glutamate transporter

92

425

426

FROMMER & NINNEMANN Table 1

(cont inued)

Rabbit o ligopeptide transporter PEPT!

45

Mouse amino acid transporter

30,97

+ Rat kidney Na SO cotransporter

III

Rabbit sodium/phosphate cotransporter 2 Bovine cyclic nucleotide-gated Ca + channel

190

Rat kidney water channel

62

Rat gap junction proteins 2+ Bovine Ca sensing receptor

40

Mulitple neurotransmitter transporters

reviewed in 9


�

192

26

Multiple mammalian receptor proteins

reviewed in 166

Na+jH

187

Anion channels

reviewed in 89

Potassium channels

reviewed in 86

Expression of membrane proteins in insect cells Arabidopsis K+ channels AKTI/KAT!

RHedrich&H

Sentenac, unpublished results

Functional expression of membrane proteins in COS cells Mammalian CD2/CD28

7

Plasmodium surface antigen

43

Mammalian interleukin receptors

67, 168, 194

Vacuolar adhesion molecule

133

Mouse receptor kinase

114

targeting to the plasma membrane and fully complemented pmal. The same yeast mutant was used to compare the biochemical properties of the three known major H+-ATPase isoforms (135). Yeast expression was also used to demonstrate that the membrane-spanning subunit of the vacuolar pyrophos phatase is sufficient for proton translocation (96, 136). Organellar membrane proteins have also been functionally expressed in yeast. Although mitochon drial porins from potato expressed in yeast can be functionally reconstituted in lipid bilayers, only one of the isoforms is able to complement the respective yeast mutant (74; L Heins & UK Schmitz, unpublished data). In the case of plastids, yeast expression helped to resolve the debate over whether the major protein of the inner envelope of chloroplasts serves as the triose phosphate trans locator (TPT) or as the import receptor for nuclear-encoded proteins. Expression, affinity purification from yeast, and subsequent reconstitution of the tagged protein in membrane vesicles demonstrated that TPT functions in triose phosphate transport in the same way that it functions in chloroplasts and that it is targeted to internal membranes (108).


Table 2

Expression of plant proteins in heterologous hosts

Functional expression in yeast

Reference

Wheat a-amylase

152

Tomato ethylene forming enzyme EFE

70

Rye SNF1 kinase homologue RKIN1

3


Tomato GTP-binding protein RAN Arabidopsis GTP-binding protein RAB6

14

Arabidopsis protein phosphatase 1 (PPIA-AT)

46

Arabidopsis type I protein phosphatase homologue

129

Tobacco protein kinase homologue NPK

10

Alfalfa cdc2 homologues

78

Maize mitochondrial T-URF 13

65

Maize TATA binding proteins (TBP)

184

Plant transcription factor TGAI

154

Maize transcriptional activator Opaque-2

117

Potato sucrose synthase

145

Functional expression of plant transport proteins in yeast Chlorella g lucose transporter HUP1

156

Arabidopsis glucose transporter STPI

157

Spinach triose phosphate translocator

108

Potato mitochondria porins

74

Arabidopsis H+-ATPases

135

Arabidopsis vacuolar pyrophosphatase subunit

96

Cloning of plant genes by complementation in yeast Arabidopsis chorismate mutase

41

Rape chloroplast 3-isopropylmalate dehydrogenase

42

Potato ATP-sulfurylase

99

Arabidopsis sterol synthesis cyc1oartenol synthase

33

Arabidopsis homologues of 8 auxotrophic mutants

123

URAi ,2,4-JO; ADE2, HiS3, LEU2, TRPi Arabidopsis homologue to Sec12 ER type II protein 36

+ Arabidopsis K -channel KAT l

4

Arabidopsis K +-channel AKTl

165

Wheat K+jH+ symporter HKTI

159

Plant sucrose transporters SoSUTl , StSUT l

144, 145

Arabidopsis amino acid transporter AAPI-5,

49,56,82, 103

NAT2 Arabidopsis peptide transporters AtPTRl-2,NTR1

58,174

Arabidopsis ammonium transporter AMTI

128

Arabidopsis amino acid transporter AATl

59

427

428

FROMMER & NINNEMANN

Table 2

(continued)


Cloning by suppression of phenotype in yeast Arabidopsis kinase

173

Arabidopsis kinase (amino acid transport)

M Kwart& WB Frommer, unpublished data

Potato kinase (phosphate transport)

G Leggewie, L Willmitzer & JW Riesmeier, unpublished data

Potato zinc finger protein (sucrose uptake)

Kiihn& WB Frommer, unpublished data

Arabidopsis catalase (amino acid transport)

S Delrot, personal communication

Arabidopsis hydroxymethyl CoA synthase (amino

S Delrot, personal communication

acid transport) Functional expression of plant membrane proteins in oocytes Chlorella glucose transporter HUPI

6, 22

Arabidopsis hexose transporter STPI

22, 23

Arabidopsis vacuolar water channel y-TIP

115

Arabidopsis maize and potato K+-channels

27, 1 25, 158

Arabidopsis nitrate transporter CHL l

182

Spinach sucrose transporter SoSUTl

KJ Boorer & E M Wright, personal communication

Arabidopsis amino acid transporters AAP/NAT

KJ Boorer& EM Wright, personal communication

Arabidopsis plasma membrane water channels

90

(PIP) Expression cloning in mammalian cells Arabidopsis plasma membrane water channels

90

(PIP)

Isolation o/Transporter Genes The major breakthrough in transport physiology was the isolation of carrier genes involved in the uptake and distribution of specific nutrients. Two differ ent potassium channels and a high-affinity K+/H+ symporter were isolated by transformation of yeast potassium-uptake mutants (4, 159, 165). Similarily, a new family of amino acid permease (AAP) genes was isolated using a proline uptake-deficient yeast strain (58, 103). The AAPs are high-affinity amino acid transporters with broad substrate specificity. Complementation of a yeast mu tant deficient in histidine uptake and metabolism led to the identification of additional members of this family (49, 82, 103). A low-affinity amino acid transporter related to the mammalian counterpart and a protein (NTR1) related to the family of nitrate and peptide transporters were also identified (57, 59). To date, ten different amino acid transporters have been isolated by yeast expression cloning (WB Frommer, unpublished data). A plant peptide trans porter, PTR2, which is related to NTRl, was able to complement a yeast mutant deficient in peptide uptake (102). Because mammalian multi-drug


429


resistance proteins (MDRs; 174) can complement the ste6 mutation in yeast, they were used to isolate plant homologues (L Covic & RR Lew, personal communication). Several genes affecting salt tolerance were identified in yeast. However, the use of these yeast sodium efflux mutants did not allow the identification of plant genes that encode efflux systems (63; A Rodriguez Navarro, personal communication).

Extending the Scope of Complementation Genetically well-characterized fungi such as Neurospora crassa and Aspergil Ius nidulans (138) provide a rich source of mutants. Sulfate transporters have been cloned from N. crassa by homologous complementation (95). These N. crassa genes were then used to isolate respective genes from S. cerevisiae and to construct a yeast deletion mutant. Selection of this mutant on low sulfate allowed the isolation of plant genes by complementation (169, 170; FW Smith, PM Ealing, M Hawkesford & DT Clarkson, personal communication). Het erologous complementation of a yeast ammonium-uptake mutant has led to the isolation of high-affinity ammonium transporter genes from plants (39, 128). An extension of the method is to modify metabolic pathways in yeast for constructing strains suitable for complementation. Despite intense biochemical attempts, the major sugar carrier of plants were refractory to analysis. Yeast complementation seemed impossible because S. cerevisiae secretes an inver tase that hydrolyzes the sucrose extracellularly and takes up the products by efficient hexose transporters. To circumvent this problem, an invertase-defi cient mutant was constructed that functionally expressed a sucrose synthase that enabled the mutant to metabolize sucrose intracellularly. This strain was used in a manner analogous to classical mutant systems for complementation cloning of a sucrose transporter gene (144, 145). Analysis of yeast cells expressing the transporter indicated a proton symport mechanism and a sug gested role in phloem loading for the cloned sucrose transporter. Direct evi dence for the function of this protein comes from transgenic plants in which expression of the sucrose transporter was partially inhibited by antisense RNA (146).

Applications for Structure-Function Studies After new genes are cloned by complementation, the biochemical properties of the proteins can be studied directly. The expression assay also allows analysis of structure-function relationships. A classical method in bacterial and fungal genetics is to select mutations from random mutagenized DNA rather than creating alterations by site-directed mutagenesis. Functional expression of heterologous proteins makes this technique applicable to many genes. For example, the use of a toxic glucose analogue as a selective agent allowed the identification of membrane-spanning domains of the glucose transporter that


430

FROMMER & NINNEMANN

are relevant for substrate affinity and that may be involved in substrate recog nition (193). Studies on membrane transport processes in yeast and animal cells indicate that these processes are highly regulated (8, 68). Regulation acts at different levels, from targeting to the membrane to modification of protein activity and degradation of the gene products (lOa, 107). Heterologous complementation may allow isolation of regulatory plant genes in the relevant yeast mutants.

Heterologous Promoters in Yeast Functional complementation with genomic libraries from animals has shown that heterologous promoters can function in yeast. Some plant and plant-virus promoters also function in yeast, and a high degree of conservation has been found for gene regulatory mechanisms (155). For example, the transcriptional regulator for maize zein proteins encoded by Opaque-2 can substitute for the nitrogen starvation gene GCN4 in yeast (117). Additional experiments are necessary to study these similarities in more detail.

Requirements for Heterologous Complementation of Mutations in Yeast QUALITY OF THE MUTANT For many yeast mutants used for complementation, the molecular basis of the mutation is not clear. The reversion frequency of point mutations can lead to large numbers of false positives in the selection. To circumvent these complications, gene disruption should be used to construct stable mutants in the gene of interest (152).

The principles of targeting seem to be conserved between organisms (17). The examples given above may indicate that many heterologously expressed pro teins are targeted correctly in yeast. Although it seems probable that the plant proteins identified by complementation of yeast plasma-membrane transport mutations are targeted to the correct compartment, direct evidence for localiza tion in the plant plasma membrane is required. Yeast targeting sequences can be used to improve heterologous expression (125). Only some of the plant proteins that complement the yeast functions are directly homologous to their yeast counterparts. Ammonium and peptide transporters as well as the amino acid transporter AAT1 from plants are related to their yeast counterparts. In contrast, no homologues of the plant AAP-amino acid permeases and the potassium transporters have been found in yeast. Thus sequence homology does not seem to be crucial for the targeting. Interestingly, if the heterologous proteins lack sequences required for assembly or targeting to the correct compartments in the cell, mistargeting can occur (135). For example, a plasma membrane gap ROLE OF TARGETING AND STRUCTURE FOR FUNCTIONAL EXPRESSION



431

junction proteolipid from arthropods functionally complements a yeast mutation in a vacuolar proteolipid gene because of incorrect targeting (80). Evidence has been presented that, in yeast, the tonoplast serves as a default compartment when targeting fails (32, 148). If true, then plant proteins localized to the plasma membrane of yeast are likely to be localized on the same membrane in plants. In standard expression experiments, only a single cDNA is expressed. This does not allow the identification of genes encoding polypeptides that are subunits of multimeric proteins or that are dependent on specific cofactors. The correct folding of monomers and the subsequent assembly into oligomers often represents a prerequisite for protein transport from the ER (135). Mis folded or unassembled proteins tend to accumulate in the ER or are rapidly degraded. When factors necessary for the proper assembly of heterologous proteins are lacking or different in yeast, incorrect targeting can occur. The four subunits of the pentameric nicotinic acetylcholine receptor (a2I3Yo) were coexpressed in yeast (87). The hydrophobic polypeptides entered the secretory pathway, where they were processed and glycosylated. However, in contrast to oocyte expression, no functional receptor was detected in yeast, possibly be cause of improper folding or assembly. Because of our detailed knowledge of targeting processes and the large set of mutants in the protein secretion pathway, yeast complementation might provide a tool to isolate plant homologues. However, despite multiple trials, few cases of successful complementation of these processes have been re ported (105a). Indications that the interaction domains in the multiprotein complexes are not sufficiently conserved between yeast and plants come from experiments in which a vacuolar targeting mutant could not be rescued by plant homologues. When the nonconserved C-terminal domain of the plant protein was replaced by the yeast counterpart, the chimeric protein was able to rescue the mutant (189). Alternative approaches have been developed to iden tify proteins that interact with a protein for which the gene has already been cloned. This two-hybrid system has been used to identify a number of genes such as DNA-binding proteins and kinases (47). TOXICITY OF THE GENE PRODUCT FOR THE HOST CELL Because the initial step for complementation of yeast mutations is the construction of a cDNA library in E. coli, the potential toxicity of proteins to E. coli can represent a problem. Toxic effects range from mildly deleterious to completely lethal (151). Expression of integral membrane protein genes can be toxic for E. coli (160). The toxicity may lead to elimination of certain genes during amplification of the cDNA library. The use of the shuttle vector pFL61 (123) provides an alternative because the vector prevents background expression from the cloned cDNAs in E. coli. For further subcloning, E. coli vectors with low plasmid copy number are recommended (0 Ninnemann & WB Frommer, unpublished data).

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YEAST AS A SOURCE FOR PURE PROTEIN Affinity purification of functionally expressed tagged proteins is a simple method in principle (l 08, 175). Yet, one of the major disadvantages of yeast expression is the comparatively low yield. Frequently this results from low transcription of foreign genes (149).

SUPPRESSION OF THE MUTANT PHENOTYPE Another complication can be ge netic suppression, i.e. the phenomenon in which a gene functionally different from the mutation to be complemented can mimic functional complementation. Extracellular hydrolysis of substrates that cannot be taken up by the mutant strain, and subsequent uptake of the products by different carriers, can mimic functional complementation of a transport mutant. Chaperonin-like proteins from humans were found to complement a yeast histidine transport mutation (164). The chaperonin relieved the nitrogen repression of the yeast endogenous general amino acid permease, GAP, probably by stabilizing a GAP-activator protein. Complex selection schemes may also be sources for artifacts. Mutations in a glucose transporter gene can suppress potassium-uptake deficient mutations in yeast (5). Several other examples exist of cloned suppressor genes that complement different transport mutations (Table 2). Complementation of the putative branched-chain amino acid transport mutant, BAP l , is based on the presence of the toxic compound sulfometurone (39a). Attempts to complement the mutant with a plant cDNA library did not allow identification of amino acid transporters; instead, detoxifying enzymes such as catalase and hydroxymethyl CoA synthase were identified (S Delrat, unpublished data). Finally, we and others have observed artifactual complementation by complex plasmid rearrangements or chimeric cDNAs (124).

EXPRESSION OF FOREIGN GENES IN ANIMAL CELLS

Transient Expression in Xenopus laevis Oocytes The X. laevis oocyte has become a major heterologous expression system because of its large size (34, 53, 69, 153, 160). Both soluble enzymes and integral membrane proteins have been expressed or cloned in oocytes. Some examples of transport proteins are shown in Table 2. Oocytes are ideal for studying transport processes because they are amenable to tracer-uptake stud ies and electrophysiological analysis. Similar to yeast, oocytes are frequently used to functionally characterize genes isolated by other means. The oocyte can also be used to identify unknown genes if suitable screening systems, such as uptake of radioactive tracers, are available. Alternatively, changes in ion flow can be determined by two-electrode voltage-clamp studies, provided the substrate is charged, cotransports a charged ion, or induces endogenous cur rents upon uptake. This technique is efficient, because transport can be meas-



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ured with mixtures of substrates or substrates that are difficult to label. A major disadvantage of transport studies in yeast cells is the problem of main taining the membrane potential during uptake experiments. In oocytes, tracer and clamp experiments can be combined, thus allowing the maintenance of membrane potential even if substrate uptake leads to depolarization of the membrane. Oocyte expression has been used widely for animal gene research; how ever, in recent years it has been extended to the plant realm, demonstrating the general feasibility of this approach for cloning (162). The general approach of cloning by oocyte expression includes the following steps: (a) Capped rnRNA is synthesized in vitro from a cDNA library. (b) RNA is injected into oocytes. (c) Tracer or voltage-clamp studies are used to detect the activity of interest. (d) RNA or cDNA library is fractionated stepwise, pools are injected sepa rately, and this step is repeated until a single cDNA is identified. Oocytes had been used successfully both for expression of cloned genes (69) and to isolate genes with novel functions (130). The Na+/glucose sym porter was isolated using 14C tracer studies (72). Many channels and receptors were identified subsequently by electrophysiological approaches (54, 79, 179). G-protein-coupled receptors have been detected indirectly as a result of in creasing intracellular calcium levels that activated calcium-dependent chloride currents (53, 113). Using a screening procedure with radiolabeled sulfate and phosphate, investigators were able to isolate genes for these carriers from kidney (111, 190). The same approach allowed the isolation of mammalian peptide transporters (45). The identification of other proteins, such as neuro transmitter transporters, has been reviewed elsewhere (9). The cellular function of a mammalian membrane protein acting as a retrovirus receptor was demonstrated by an elegant electrophysiological study of oocytes expressing the protein. The addition and fractionation of a complex mixture of substrates provided evidence that the protein is an amino acid transporter (97). Further analysis allowed the study of glycosylation, structure function relationship, and the facilitated diffusion mechanism for transport (30, 98). Despite the efficient use of this system for other organisms, no plant protein has been isolated by expression cloning in oocytes.

Characterization of Plant Transporters in Oocytes In the case of plant transporters, oocytes have been used mainly as a tool to characterize electrophysiological properties. Such measurements are possible in yeast but do not yet represent a standard technique (18). Based on the finding that mammalian sodium-dependent glucose transporters could be char acterized electrophysiologically in oocytes, the plant H+ /glucose transporter STPI was expressed and studied in oocytes (6, 22, 23). The A. thaliana KAT1 gene that was isolated by yeast complementation was shown to encode an


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inwardly rectifying potassium channel (73, 158, 165). The same holds true for a KATl homologue predominantly expressed in guard cells that was identified by heterologous screening of a potato epidermal cDNA library (125). A de tailed analysis in oocytes demonstrated that the potassium transporter HKTl functions as a high-affinity K+/H+ symporter (159). Expression in oocytes showed that the A. thaliana Chll protein, which is responsible for chlorate resistance, is involved in low-affinity nitrate transport (182). The stoichi ometry for proton cotransport of the A. thaliana sucrose transporter and AAPs has also been analyzed (KJ Boorer & EM Wright, unpublished data). The use of oocytes has allowed researchers to determine the function of integral mem brane proteins of the tonoplast. Oocytes showed an increased water permeabil ity when expressing the plant vacuolar y-TIP, demonstrating a function in water transport (115). The same could be demonstrated for glucose trans porters and for TIP-related plasma membrane proteins from plants and animals (48, 90, 197). In yeast, substrate specificity can be analyzed easily by simple growth assays, by inhibition studies, or by radioactive-uptake measurements. Because radiolabeling of compounds can be difficult, substrate specificity is often determined by competition studies. The results are of limited value because substrates cannot be distinguished from inhibitors. Direct determination of uptake in oocytes by electrophysiological assays normally does not allow differentiation between inhibitors and nonsubstrates. This differentiation can be achieved either by competition assays or by studying pre-steady-state cur rents (109, 110). Such an analysis is currently used to determine substrate specificity and, in combination with site-specific mutagenesis, to elucidate structure-function relationships of sugar transporters from animals and plants (EM Wright, personal communication). A limitation of oocytes is that measurements are performed with living cells in which regulatory and metabolic events can affect the function of the heterologous protein. Internal perfusion of the oocyte during transport studies circumvents this problem by decoupling the memb!ane from metabolism, leaving basically an in vitro system (20, 178). Analogous to the finding in yeast that heterologous expression of soluble proteins can mimic transporter expression, amino acid transport activities in oocytes can be induced by sol uble proteins that may activate endogenous transporters (19, 180, 188). These proteins may represent regulatory subunits (88). One of the advantages of oocyte expression is that proteins with multiple subunits can be identified by simultaneous expression of whole or fractionated cDNA libraries. After cloning and expression of one subunit, subsequent rounds of screening can be used to identify other subunits by looking for modulated activity (88). Coexpression in oocytes can also be used to study regulation, e.g. the activation of cloned K+ channels by G-proteins (143). Such



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approaches will help researchers to characterize regulatory networks and to obtain a better understanding of signal transduction pathways. The handling of oocytes is certainly more complicated than yeast cells. Efficient expression requires permanent access to high-quality oocytes. Fur thermore, oocytes are not suitable for all purposes, e.g. they do not contain plastids or vacuoles. The finding that plant vacuolar proteins are functional at the oocyte plasma membrane indicates that the plasma membrane serves as the default compartment .in these cells, thus extending the scope of the method (32, 148).

Expression in Insect Cell Cultures Baculoviruses are valuable for producing preparative quantities of foreign proteins. Besides the high yield of protein (up to 150 mg/L for membrane proteins) (91), the proteins are posttranslationally modified. As in the other expression systems, several proteins may be expressed simultaneously (52). The utilization of baculovirus as a vehicle for protein expression for both soluble and integral membrane proteins has been demonstrated repeatedly, mainly for mammalian and viral genes (52). This method has allowed func tional expression and electrophysiological characterization of a human K+ channel (91). In addition, several soluble proteins of plant origin have been expressed: storage proteins, proteases (91), histidinol dehydrogenase (126), and mitochondrial proteins (101). The A. thaliana KATl potassium channel showed similar properties when expressed in insect cells and in oocytes (64, 73, 158; F Gaymard & H Sentenac, unpublished data; I Marten & R Hedrich, unpublished data). Functional expression of another A. thaliana K+ channel, AKTl, in oocytes has been unsuccessful so far, but high quantities of pure protein have been isolated by expression in insect cells (H Sentenac, personal communication).

Expression in Mammalian Cells Expression and cloning of genes in mammalian cell cultures has contributed to our understanding of signaling processes. Plasmids containing the SV40 origin of replication can be used for transient expression of heterologous genes in mammalian cell lines (7). Expression can be detected by immunoscreening, by using radiolabeled ligands, by phenotypic screening, or by complementation of mutants (133). A modification of the detection system allows the identifica tion of intracellular proteins by immunostaining of permeabilized fixed cells (119). This expression system has led to the identification of many receptors and cell surface proteins (Table O. COS and other cell lines have been used successfully (29, 94). Mammalian cell lines can also be used for expressing plant proteins. Expression of a cDNA library from A. thaliana roots in cell cultures and subsequent screening with antisera directed against the plasma

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membrane enabled the identification of plasma membrane water channels

(90). In contrast to research in mammals, very few plant receptors have been identified at the molecular level (84). Expression in mammalian cells appears to represent an excellent tool to isolate new receptor genes and should help to overcome this lag. Furthermore, ectopic expression of foreign genes can be


used to study defined physiological questions (130, 139).

EXPRESSION OF GENES IN PLANT CELLS Heterologous expression in yeast and animal cells is well established. Surpris ingly, plants or plant cells have not been used for this purpose, even though the technology is available. Well suited for this purpose are unicellular organisms such as green algae, for which transformation protocols have been developed.

A number of mutants, e.g. those involved in nitrogen uptake and reduction, have been described that could be used for complementation (51, 141). Proto plasts or cell cultures from higher plants can be used as transient expression systems with transformation rates of up to 60% (0 Neuhaus, personal commu nication). Even stable transformation rates for protoplasts are high enough for some species for complementation approaches (186). Several cell lines are available from different plants [e.g. tobacco BY-2 cells, which can be synchro nized (127)]. Expression in plant cells would be particularly advantageous for resolving plant -specific problems, such as the targeting of vacuolar or plastidic proteins. Correct targeting of transiently expressed genes to chloroplasts was demonstrated by immunofluorescence (121). The feasibility of such ap proaches has been shown by expression of genes in higher plants with genetic backgrounds that lack a certain function, e.g. the demonstration that patatin encodes an esterase activity (150). Finally, ectopic expression of genes from heterologous species has proved to be an efficient tool for studying plant physiology (185).

CONCLUSIONS Heterologous expression systems are powerful tools for isolating new genes and for characterizing proteins from all organisms. The major expression system for plant genes is yeast, which has allowed the isolation of more than

20 transporter genes. These genes represent only a small fraction of the trans porters present in plants. Heterologous expression of plant genes in systems other than yeast will be important in the future. Combinations of the different expression systems-i.e. oocytes for electrophysiological characterization, yeast for the selection of mutant proteins with altered functions, and cell cultures for the production of protein for crystallization-will allow a better understanding of how plants and animals function.


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ACKNOWLEDGMENTS We are very much indebted to all those who have provided us with unpub lished data. We want to thank especially Michele Minet and Julian Schroeder for helpful discussions. In this context we would like to thank Jorg Riesmeier whose work was crucial for establishing yeast complementation in our group.


We also would like to thank Doris Rentsch, Nicholas Provart, Frank Lauter, and Remi Lemoine for critical reading of the manuscript. Any Annual Review chapter, as well as any article cited in an Annual Review chapter, may be purchased from the Annual Reviews Preprints and Reprints service. 1·800·347·8007; 415·259·5017; email: [email protected]

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