Genetic Approaches to Electrogenic Proton Transport by a Yeast H+-

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14 Genetic Approaches to Electrogenic Proton Transport by a Yeast H -ATPase +

D . S. Perlin , D . Seto-Young , B. C . M o n k , S. L . Harris , S. N a , and J. E. H a b e r 1

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Department of Biochemistry, The Public Health Research Institute, New York, NY 10016 Department of Biology, Brandeis University, Waltham, MA 02254

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A genetic approach is used to probe electrogenic proton transport by the Saccharomyces cerevisiae plasma membrane H -ATPase (PMA1). Mutations in PMA1 , selected by growth resistance to hygromycin B, cause a depolarization of cellular membrane potential. Enzyme from one mutant, pma1-105 (Ser368 -> Phe), is largely insensitive to im posed membrane voltage and appears to catalyze electroneutral H transport. A countercurrent of K may provide charge compensation in this enzyme. Second-site mutations that partially revert the pma1105 phenotype were identified within the membrane sector and pro vide strong evidence for coupling between cytoplasmic and membrane domains. Mutations conferring hygromycin Β resistance have been identified in both cytoplasmic and membrane domains, and multiple transmembrane segments are presumed to be important for electro genic H transport. However, a special emphasis is placed on the role of transmembrane segments 1 and 2, which provide the starting point for a more inclusive structural model of the membrane sector. +

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Approaching a Proton Transport Mechanism E n z y m e - l i n k e d p r o t o n transport systems have recently b e c o m e the focus o f intense molecular scrutiny. S u c h studies have established some o f the g r o u n d w o r k necessary to investigate long-standing mechanistic questions: (1) C a n a translocation mechanism that moves H

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b e defined at the molecular level?

(2) C a n such a m e c h a n i s m accommodate the transport o f other ions like N a , +

0065-2393/94/0235-0315$08.00/0 © 1994 American Chemical Society

Blank and Vodyanoy; Biomembrane Electrochemistry Advances in Chemistry; American Chemical Society: Washington, DC, 1994.

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BIOMEMBRANE ELECTROCHEMISTRY

K , a n d C a ? T o address these questions properly requires a better understanding o f the three-dimensional structure o f transport enzymes, the identification o f p r i n c i p a l amino acids that participate i n the translocation reaction, and an appreciation of the p r o t e i n molecular dynamics that con tribute to i o n b i n d i n g (and release) a n d movement i n the translocation domain. +

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F e w examples exist where the molecular architecture of a transport protein can be described at h i g h resolution ( i , 2). Difficulties arise because o f the amphipathic character and preference o f m e m b r a n e proteins for a hydrophobic environment. These properties make the growth of suitably diffracting three-dimensional crystals difficult (3). T h e difficulties inherent i n visualizing these structures at h i g h resolution have p r o m p t e d most investiga tors to rely o n theoretical predictions for membrane p r o t e i n disposition (4) and structure (5). M o d e l s d e r i v e d f r o m theoretical considerations are gener ally two-dimensional topographic maps that readily predict the sequences of hydrophobic amino acids that he w i t h i n the membrane but fail to predict the spatial relationships o f individual amino acids or protein structure elements. Nonetheless, this m o d e l i n g approach can be an extremely valuable tool i n targeting putative amino acids that may participate i n the transport reaction. T h e application o f site-directed mutagenesis for investigating amino a c i d functionality has p r o v i d e d a significant advance i n attempts to probe the molecular basis o f p r o t o n translocation. N u m e r o u s studies that encompass diverse p r o t o n transport systems have p r o v i d e d evidence for the role of m e m b r a n e - e m b e d d e d charged amino acids i n the transport reaction. These amino acids i n c l u d e G l u , A s p , L y s , A r g , a n d other residues capable o f a c i d - b a s e reactions such as H i s and T y r . I n several systems, models to describe localized proton b i n d i n g and release have e m e r g e d (2, 6, 7), but a generalized transport scheme has not b e e n forthcoming. T h e essential role of p r o t e i n structure dynamics as a mechanistic consid eration i n p r o t o n transport has focused attention o n amino acids such as proline (8, 9), w h i c h can confer localized flexibility w i t h i n a n d between helical segments. E x c e p t for the unique case o f bacteriorhodopsin, w h i c h is amenable to spectroscopic examination (10), little is k n o w n about the nature of molecular dynamics i n transport enzymes.

The Yeast H-ATPase as a Model System T h e plasma m e m b r a n e H - A T P a s e f r o m Saccharomyces cerevisiae is a highly electrogenic proton p u m p that maintains cellular membrane potentials i n excess of —200 m V . T h i s enzyme is used as a m o d e l system to probe the mechanism of electrogenic p r o t o n transport because it is readily amenable to genetic, biochemical, and biophysical analysis. T h e H - A T P a s e plays an essential role i n intracellular p H regulation a n d maintenance o f electrochemi+

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Electrogenic Proton Transport

cal p r o t o n gradients across the plasma m e m b r a n e ( I I ) ; i t belongs t o t h e P-type family o f ion-translocating A T P a s e s that f o r m acyl phosphate i n t e r m e diates d u r i n g catalysis a n d are sensitive to i n h i b i t i o n b y vanadate (12). T h e gene e n c o d i n g the H - A T P a s e , PMA1, has b e e n c l o n e d a n d sequenced (13) and subjected to extensive mutagenesis for the purpose o f functional m a p p i n g (14, 15). A h i g h degree of amino a c i d sequence a n d structural homology has +

b e e n observed w i t h H - A T P a s e s f r o m other f u n g i a n d plants, as w e l l as for more distantly related enzymes like the N a - K - A T P a s e a n d C a - A T P a s e (13, 16). T h e H - A T P a s e can be p u r i f i e d a n d reconstituted i n liposomes, a n d its electrogenic transport properties characterized i n vitro (17). These fea tures make the yeast H - A T P a s e a n attractive enzyme f o r t h e study o f charge-transfer reactions. +

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T h e isolation o f pmal mutants that are resistant to the aminoglycoside antibiotic hygromycin Β (18) has p r o v i d e d t h e opportunity to probe the charge-transfer process i n t h e H - A T P a s e . These h y g r o m y c i n Β resistant cells have a defect i n the H - A T P a s e that causes a generalized depolarization of cellular m e m b r a n e potential (19). T h i s chapter w i l l discuss h o w r a n d o m mutations i n PMA1 may help identify putative transmembrane structures participating i n electrogenic p r o t o n transport. +

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Genetic Probing of Electrogenic Proton Transport G e n e t i c approaches t o analysis o f molecular structure a n d function can b e d i v i d e d into two types. N o n r a n d o m site-directed mutagenesis targets specific amino acids a n d is most successfully a p p l i e d w h e n elements o f structure are reasonably w e l l understood. T h e more classical, r a n d o m mutagenesis a p proach does n o t require assumptions to b e made about essential residues. R a n d o m mutagenesis has the advantage that numerous a n d diverse function ally defective mutants can b e isolated. T h i s latter approach was selected for initial probe studies o f PMA1. R a n d o m mutagenesis r e q u i r e d a special selection procedure to address the p r o b l e m that the H - A T P a s e is essential for c e l l growth a n d gross perturbation of enzyme function is lethal to the cell. A positive selection m e t h o d for isolating pmal mutants was developed based on growth resistance o f cells to the aminoglycoside antibiotic h y g r o m y c i n B . T h e initial screen generated a collection o f 5 3 pmal mutants w i t h a w i d e range o f cellular phenotypes i n c l u d i n g sensitivity to l o w external p H , weak acid loading ( 0 . 1 - M acetate, p H 5.0), a n d N H (18). A l l o f the mutant enzymes characterized were f o u n d to have kinetic defects that decreased the m a x i m u m initial velocity ( V ) o r the M i c h a e l i s - M e n t e n constant (K ) for A T P hydrolysis, a n d several mutant enzymes w e r e insensitive to vanadate-ind u c e d i n h i b i t i o n (15). T h e cellular basis for growth resistance to h y g r o m y c i n Β was experimentally d e t e r m i n e d t o b e a deficiency i n the maintenance o f the n o r m a l hyperpolarized m e m b r a n e potential i n yeast (19). This potential +

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m a x


m

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has b e e n estimated to exceed —200 m V ( 2 0 ) . Because direct electrophysio logical examination o f yeast is not possible, the l i p o p h i l i c distribution probe tetraphenylphosphonium ( T P P ) was used as a representative measure o f cellular m e m b r a n e potential. F i g u r e 1 illustrates that mutant cells metaboliz i n g glucose take u p 2 . 5 - 1 0 - f o l d less T P P than w i l d - t y p e cells, w h i c h suggests that pmal mutant enzymes were defective i n maintaining the normally h i g h level o f m e m b r a n e potential.

A Proton Transport Dilemma T h e m e m b r a n e potential depolarization i n pmal mutants c o u l d be explained i f the mutant enzymes were less active i n p u m p i n g protons across the m e m b r a n e . T h i s notion was supported b y kinetic studies o n A T P hydrolysis b y these enzymes that showed small but significant decreases i n V (15). H o w e v e r , a d i l e m m a arose w h e n whole c e l l m e d i u m acidification experi ments were p e r f o r m e d , w h i c h reflect the action o f the H - A T P a s e i n vivo. T h e rate o f glucose-induced p r o t o n efflux b y pmal mutant cells was f o u n d to be considerably better than that o f wild-type cells ( F i g u r e 2). O n l y w h e n h i g h external K was i n c l u d e d i n the m e d i u m to m i n i m i z e differences i n m e m brane potential between mutant a n d w i l d - t y p e cells d i d the activity o f the m a x

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TIME,

min

Figure 1. Uptake of [ C]TPP by p m a l mutants. The uptake of [ C]TPP in the presence of glucose was determined for wild-type (A), pmal-101 pmal-105 (U), pmal-114 (m), pmal-141 (w), pmal-147 fa), and pmal-155 (O) cells. The reaction was initiated by the addition of [ C]TPP to the cell suspensions. (Reproduced with permission from reference 19. Copyright 1988 American Society of Biochemistry and Molecular Biology.) 14

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Blank and Vodyanoy; Biomembrane Electrochemistry Advances in Chemistry; American Chemical Society: Washington, DC, 1994. •

, u

pmai-114 i

glu PMA1 1

glu

Figure 2. Effect of KCl on medium acidification. Wild-type (PMA1) and p m a l mutant cells (pmal-109 and pmal-114) were analyzed for glucose-induced medium acidification. (Reproduced with permission from reference 19. Copyright 1988, American Society for Biochemistry and Molecular Biology.)

pjmajM09

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wild-type approach that o f the mutants ( F i g u r e 2). T h e s e results suggest that a simple kinetic explanation is inadequate to account f o r the m e m b r a n e potential defect i n pmal mutants. C l e a r l y , some other fundamental property o f electrogenic p r o t o n transport b y the pmal mutant H - A T P a s e h a d b e e n altered. +

Electrogenicity by the H -ATPase +

T o assess more directly w h e t h e r the electrogenicity o f the H - A T P a s e was altered, an i n vitro assay was developed to examine the activity o f the enzyme as a function o f a p p l i e d voltage. T h i s approach was necessary because previously it has not b e e n possible to use conventional electrophysiology o r patch-clamp techniques to study the c u r r e n t - v o l t a g e behavior o f the H A T P a s e i n yeast. A simple system was used to examine the effects o f interior positive m e m b r a n e potential formation o n the catalytic properties o f p u r i f i e d H - A T P a s e reconstituted into proteoliposomes. I n this system, electron flow f r o m vesicle-entrapped ascorbate to external K F e ( C N ) is mediated b y the l i p o p h i l i c electron carrier tetracyanoquinodimethane ( T C N Q ) ( F i g u r e 3). Interior positive m e m b r a n e potential formation was readily f o l l o w e d using the optical probe oxonol V ( F i g u r e 3) a n d c o u l d be collapsed either b y the addition o f permeant anions like S C N ~ o r b y the addition o f valinomycin to K - l o a d e d vesicles ( F i g u r e 3). Potentials w e r e calibrated b y a n u l l - p o i n t titration assay utilizing K a n d v a l i n o m y c i n - i n d u c e d diffusion potentials (17). A m a x i m u m m e m b r a n e potential o f approximately 254 m V was f o u n d to reduce A T P hydrolysis b y w i l d - t y p e enzymes u p to 4 6 % . T h e sensitivity o f the enzyme to inhibition b y vanadate was also m o d i f i e d u n d e r these c o n d i tions (17). T h e relationship between A T P hydrolysis a n d membrane voltage is shown i n F i g u r e 4. T h e w i l d - t y p e enzyme is relatively insensitive to voltage over approximately 100 m V , b u t t h e n shows voltage-sensitive behavior. A simple nonlinear extrapolation o f the data i n F i g u r e 4 yields a p r e d i c t e d reversal potential o f approximately 375 m V , w h i c h is consistent w i t h electro physiological studies o f the closely related N e u r o s p o r a H - A T P a s e (21) a n d a stoichiometry o f 1 H transported p e r A T P h y d r o l y z e d ( 2 2 ) . I n contrast to the behavior o f the w i l d - t y p e enzyme, a typical pmal mutant enzyme f r o m strain pmal-105 was f o u n d to b e nearly insensitive to voltage, even at a m a x i m u m potential o f 254 m V (17). T h i s observation suggests that mutant enzyme pmal-105 is insensitive to physiological levels o f voltage. T h e s e data further suggest that other enzymes f r o m h y g r o m y c i n Β resistant pmal mutants may b e defective i n electrogenic p r o t o n transport. +

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Evidence for Ion Counterflow by Mutant H -ATPase +

Electroneutrality b y the mutant pmal-105 H - A T P a s e most likely results f r o m a compensating charge movement following electrogenic p r o t o n trans+



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Figure 3. Description of systems used to generate and measure interior positive membrane potentials. (A) A schematic diagram for the ascorbate-TCNQ-K Fe(CN) reaction. The interior positive membrane potential formation is catalyzed by the lipophilic electron carrier TCNQ, which mediates the flow of electrons from ascorbate inside the vesicle to K Fe(CN) outside. (B) Membrane potential formation in reconstituted proteoliposomes was followed by the fluorescent probe oxonol V. (Reproduced with permission from reference 17. Copyright 1991 American Society for Biochemistry and Molecular Biology.)

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140 -

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100 i

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WT#1 WT#2

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roilysî

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C- 1 — C

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« — ι — « — Γ

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Membrane

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Potential

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(mV)

Figure 4. The relationship between ATP hydrolysis and membrane potential ATP hydrolysis by purified and reconstituted H -ATPase from wild-type and pmal-105 was determined as a function of imposed membrane potential. ATP hydrolysis assays were performed for only 60 s to maintain a stable level of membrane potential. A nonlinear curve-fit routine was used to derive the extrapolated line drawn for the wild-type sample data. (Reproduced with permission from reference 17. Copyright 1991, American Society for Biochemistry and Molecular Biology.) +

port, a n d there is strong precedent for this behavior i n other P-type A T P a s e s . I n the gastric enzyme, an electrogenic H equal a n d opposite K Na

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(24).

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current c a n b e balanced b y a n

current ( 2 3 ) . I n the N a - K - A T P a s e , " u n c o u p l e d " +

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flux, w h i c h is electroneutral at p H 6.5, m a y require H

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as a counterion

Finally, the apparent electroneutrality observed for the C a - A T P a s e 2 +

f r o m cardiac sarcolemma m a y also involve a counterflow o f H

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(25). T h e

fungal H - A T P a s e has no apparent catalytic requirement for any i o n other +

than H

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, although it has b e e n shown that p u r i f i e d a n d reconstituted enzyme

f r o m S. pombe transports K

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i n an A T P - d e p e n d e n t m a n n e r (26).

a patch-clamp analysis o f h y g r o m y c i n Β resistant pmal-105 branes identified an A T P a s e - d e p e n d e n t K logical voltage range ( 6 0 - 1 2 0 m V ) . A K

+

+

Recently,

mutant m e m

channel conductance i n a physio conductance was also present i n

wild-type membranes, b u t it was not A T P activated a n d r e q u i r e d higher gating voltages ( > 1 4 0 m V ) ( 2 7 ) . T h i s w o r k raised the possibility that K transport is i n d u c e d i n mutant enzymes like pmal-105

+

a n d m a y provide the

electrical countercharge necessary for apparent electroneutrality.


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P r e l i m i n a r y evidence suggests that the n e w K conductance is directly mediated v i a the H - A T P a s e , a n d two mechanistic schemes can b e envi sioned to account for this conductance ( F i g u r e 5). K transport c o u l d involve the same translocation pathway as H (but move i n the opposite direction to H ) o r require an independent antiparallel translocation pathway. T h e r e are no data to distinguish between these two alternatives. Nonetheless, a c o m m o n translocation mechanism that c o u l d accommodate H a n d K m a y b e the most attractive because i o n selectivity c o u l d b e most easily accomplished by a single specialized gating mechanism. +

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Mapping pmal Mutations G e n e t i c m a p p i n g o f h y g r o m y c i n Β resistant mutants was p e r f o r m e d to identify specific amino acids a n d domains o f p r o t e i n structure that influence the eletrogenic behavior o f the H - A T P a s e . Mutations w i t h i n the c o d i n g region o f PMA1 m a p to either the m e m b r a n e region o r a large h y d r o p h i l i c region containing the catalytic center (15). F i g u r e 6 A shows a schematic topography m a p o f the H - A T P a s e a n d the relative position o f three p r o m i nent mutations. It c a n b e seen that G l y l 5 8 -> A s p , w h i c h partially uncouples H transport f r o m A T P hydrolysis (15), is expected to l i e w i t h i n the bilayer. T h e other two mutations, Ser368 —> P h e a n d Pro640 -> L e u , are expected to be w i t h i n the catalytic d o m a i n . This analysis is consistent w i t h the finding that these latter mutations alter the kinetics for A T P hydrolysis a n d confer relative insensitivity to the mechanistic i n h i b i t o r vanadate (15). T h e fact that a mutation i n the cytoplasmic A T P hydrolysis d o m a i n can alter the mechanistic nature o f p r o t o n transport w i t h i n the m e m b r a n e strongly supports the n o t i o n o f c o u p l i n g between these disparate domains. Recently, w e p r o v i d e d more +

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Figure 5. Schematic models for ATPase-mediated K conductance. (A) K transport occurs through the same pathway as H . (B) K transport occurs through a separate pathway from Η but is still mediated by the enzyme. +

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(A)

Ser368~>Phe Gly158->Asp Outside Membrane

Asp378 Site of Phosphorylation

ATP Binding Domain

(Β) A828S

A135S 1133F

Asp378 Site of Phosphorylation Figure 6. Diagram illustrating the membrane topography of the H -ATPase. (A) The topography of the H -ATPase was determined from analysis of hydropathy profiles (16). Amino acid substitutions representing pmal-114, pmal-105, and pmal-147 mutant enzymes are indicated with respect to previously assigned functional domains. (Reproduced with permission from reference 15. Copyright 1989, American Society for Biochemistry and Molecular Biology.J (B) Topography map showing membrane locations of second-site mutations that suppress the NH/ and low-pH sensitive phenotypes of a pmal-105 mutant (Ser368 -» Phe). (Adaptedfrom reference 28. Copyright 1991, American Society for Biochemistry and Molecular Biology.) +

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direct evidence (28) for such c o u p l i n g b y showing that second-site mutations expected to lie w i t h i n the m e m b r a n e bilayer c a n suppress the l o w p H a n d N H

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sensitive phenotype i n d u c e d b y the Ser368 -> P h e mutation w i t h i n

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the cytoplasmic catalytic d o m a i n ( F i g u r e 6 B ) . T a b l e I summarizes the existing set o f genetically m a p p e d mutations, isolated f r o m assorted mutagenesis schemes, that are b e l i e v e d to lie w i t h i n the bilayer a n d result i n hygromycin Β resistance. A n important outcome o f the genetic m a p p i n g studies is that mutations c o n f e r r i n g cellular hygromycin Β resistance arid, b y inference, a d e p o l a r i z e d cellular m e m b r a n e potential are broadly distributed w i t h i n transmembrane segments 1, 2, 3, 4, a n d 7 o f the enzyme. These data suggest that multiple transmembrane elements partici pate i n electrogenic p r o t o n transport a n d c o u p l i n g to A T P hydrolysis.

Role of Membrane-Embedded Helices 1 and 2 in Electrogenic H Transport +

A detailed characterization o f hygromycin Β resistant mutants suggests that transmembrane segments 1 a n d 2 participate i n electrogenic H

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transport

a n d c o u p l i n g to A T P hydrolysis (28). These two segments a n d the connecting t u r n represent a template for the development o f a three-dimensional m o d e l for the m e m b r a n e sector o f the enzyme a n d have b e e n m o d e l e d as a h a i r p i n loop structure that is d o m i n a t e d b y a pair o f antiparallel α helices ( F i g u r e 7). T h e h y d r o p h o b i c helical elements are j o i n e d b y a short ( 3 - 4 residues) t u r n region, w h i c h includes a polar serine residue a n d a highly charged A s p residue. T h e transmembrane loop is constrained into a single tightly p a c k e d configuration w i t h the two helices p i t c h e d at a n angle o f about 15° to f o r m a n

Table I. Hygromycin Β Resistant Mutants Mapping within the Bilayer Mutation Alal35 -> Val Ilel33 -> Fhe ' Alal35 -> Ser Ilel47 -> Met* Cysl48 - * Ser Vall57 - * P h e ' Glyl58 -> Asp a

Mutation

1

Leu298 -> P h e Leu327 -> V a l Met346 -> Val Met346 -> T h r Ala828 - * S e r ' Ser836 -» C y s

b

a

a

fl

e

Helix

Helix

c

1 2 2 2 2

û

c

c

a c

3 3 4 4 7 7

These mutations were selected as second-site suppressors of either the low p H or NH sensitive phenotype of pmal-105 (Ser368 —> Phe). They confer hygromycin Β resistance when separated form the primary-site (Phe368) mutation (28). He 133 -» Phe and Alal35 - * Ser comprise a double mutant. These mutations were selected as in footnote a. They are hygromycin Β resistant when present with the Phe368 mutation, but it is not known whether they confer hygromycin Β resistance when separated from Phe368. a

4

c


326


turn

t helix 1

t helix 2

Figure 7. Amino acid sequence and molecular model for transmembrane helices 1 and 2. A molecular model generated with the program Quanta (Polygen, Inc.) that represents transmembrane helices 1 and 2 is shown next to its corresponding amino acid sequence.

inverted V-type structure that resembles the first two helices o f a four-helix b u n d l e (29). Several o f the mutations identified i n this region are p r e d i c t e d to alter the disposition o f α helices relative to each other b y sterically influencing the close p a c k i n g o f adjacent amino a c i d side chains. T h e close packing o f amino acid side chains near the loop region suggests that muta tions i n this region w o u l d b e p o o r l y tolerated. I n k e e p i n g w i t h this notion, i t was f o u n d that a relatively modest change, A l a l 3 5 -> V a l , p r o d u c e d a d r a matic perturbation i n the enzyme function. T h i s V-structure is highly conformationally active as indicated b y the finding that site-directed mutations i n the loop region, L e u l 3 8 -> T y r a n d A s p l 4 0 -> G l u , b o t h confer growth resistance to hygromycin B . I f the h y d r o p h i l i c d o m a i n extending f r o m trans membrane segment 2 is r i g i d a n d i n contact w i t h t h e central h y d r o p h i l i c d o m a i n containing the catalytic center, t h e n it is possible to explain h o w a small perturbation i n a distant p o r t i o n o f a transmembrane segment c a n influence the catalytic behavior o f the enzyme. T h e role o f transmembrane segments 1 a n d 2 i n H (and possibly K ) transport is currently b e i n g investigated b y a comprehensive mutagenesis approach. +

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Summary and Conclusions Mutations i n the Saccharomyces cerevisiae H - A T P a s e gene, PMA1, that confer cellular growth resistance to h y g r o m y c i n Β cause a generalized depo larization o f cellular m e m b r a n e potential. T h e n o r m a l hyperpolarized m e m brane potential i n yeast is maintained b y the H - A T P a s e , a n d it is b e l i e v e d that the pmal mutations alter electrogenic p r o t o n transport b y the enzyme. E l e c t r o n e u t r a l H transport b y the mutant enzymes may involve the countertransport o f K , b u t other ions i n c l u d i n g H c o u l d participate. M o r e direct evidence is n e e d e d to c o n f i r m the role o f K as a counterion a n d to probe its putative transport m e c h a n i s m . It w i l l b e important to determine w h e t h e r H and K use the same mechanistic pathway for transport. +

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+

+

A significant outcome f r o m the mutant m a p p i n g studies was the finding that amino acid substitutions w i t h i n either the m e m b r a n e d o m a i n o r cytoplas mic catalytic d o m a i n render the enzyme defective i n electrogenic p r o t o n transport. T h e disparate environments represented b y these t w o domains provide indirect evidence f o r c o u p l i n g between A T P hydrolysis a n d p r o t o n transport. M o r e direct evidence for c o u p l i n g was p r o v i d e d b y t h e identifica tion o f second-site mutations w i t h i n the m e m b r a n e sector that partially suppress the phenotype o f a primary-site mutation, Ser368 - » P h e , located near the site o f phosphorylation i n the large central cytoplasmic d o m a i n . M u l t i p l e transmembrane segments have b e e n i m p l i c a t e d i n electrogenic proton transport a n d c o u p l i n g to A T P hydrolysis. A special emphasis was p l a c e d o n transmembrane segments 1 a n d 2. M o l e c u l a r m o d e l b u i l d i n g suggests that these segments f o r m a pair o f antiparallel α helices that are b o t h tightly c o m p a c t e d a n d highly conformationally active. It is believed that this structure c a n serve as a template f o r b u i l d i n g a m o d e l o f the entire transmembrane d o m a i n . S u c h a m o d e l w i l l provide an important experimen tal tool f o r more detailed a n d direct genetic p r o b i n g o f the H transport mechanism. +

Acknowledgments T h i s w o r k was supported b y the O f f i c e o f N a v a l Research G r a n t N 0 0 0 1 4 8 9 - J - 1 7 9 2 (to D . S. Perlin), N a t i o n a l Institutes o f H e a l t h grants G M 3 8 2 2 5 (to D . S. P e r l i n ) a n d G M 3 9 7 3 7 (to J . E . H a b e r ) , a n d N a t i o n a l Science F o u n d a t i o n G r a n t D C B 8 4 0 9 0 8 6 (to J . E . H a b e r ) .

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Genetic Approaches to Electrogenic Proton Transport by a Yeast H+-

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