The enzymology of protein translocation across the Escherichia coli plasma membrane.

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THE ENZYMOLOGY OF PROTEIN Annu. Rev. Biochem. 1991.60:101-124. Downloaded from www.annualreviews.org Access provided by University of York on 01/25/15. For personal use only.

TRA��SLOCATION ACROSS THE Escherichia coli PLASMA MEMBRANE William Wickner, Arnold J. M. Driessen,l and Franz-Ulrich HartF Molecular Biology Institute and Department of Biological Chemistry, University of California at Los Angeles, Los Angeles, California 90024-1570 KEY WORDS:

translocase, secretion, translocation ATPase, leader peptide, protonmotive force.

CONTENTS PERSPECTIVES AND SUMMARY . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . .

102

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

102

LEADER PEIPTIDES . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

103

SEC PROTEIN-DEPENDENT TRANSLOCATION . . . . . . . . . . . . . . . . . . .... . . . . . . . . . . . . . . . . . . . . . SeeB . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . SeeA ............................................................ . ....... . . .. . ....................... . SeeY/E . .... . . . . . . . . . . . .. . . . . . . ..... . . . ........ . ..... . ... . . . . . . . ... ........ . . ... . . ....... . . ... . . . . . . . .

105 105 108 109

TRANSLOCATION THAT IS SEC-INDEPENDENT . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

110

ENERGETICS OF EXPORT . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

112 112 113

The Role of iJ.ilH+ .......................... . . . ............. . . . . ...... ......... . . ...... . . . . ...... . The Role of ATP ..................................................................................

COMPARISONS WITH OTHER PROTEIN TRANSLOCATION REACTIONS . . . . . . . . Targeting and Recognition . . . . . . . . . .... . . . .. .... . . . ... ... . ... ..... . . . . . . . .. . . . . .. . . . . . . . . . . . . . . . Transloearion and Energy Requirement . . . . . . . . . . . . . . . . . ..... . . . .. .... . . . . . . .. . . . . . ..... . ... . CURRENT QUESTIONS IN BACTERIAL EXPORT . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Proeoat ...... . . . .

113 115 116 117 117

'Permanenl address: Department of Microbiology, University of Groningen. Kerklaan 30,

9751 NN Har,�n, The Netherlands.

2Permanent address: Institut fUr Physiologische Chemie, Universitat Miinchen, Goethestrabe

33, D-8000 Miinchen 2, Federal Republic of Germany.

101

0066-4154/91/0701-01 01$02. 00

Annu. Rev. Biochem. 1991.60:101-124. Downloaded from www.annualreviews.org Access provided by University of York on 01/25/15. For personal use only.

1 02

WICKNER, DRIESSEN & HARTL

Mature Domains .. ......... ..... .. ... .... .... ..... .. .... ..... ..... . ..... ...... ..... ....... .... .... Unfolding........................................................................................... Translocation Pathway........................................................................... Role of Lipid.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Role of ;jflH+ . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Folding in the Periplasm........................................................................ Role of Translation. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Regulation..........................................................................................

117 118 118 119 119 119 120 120

PROSPECTUS.........................................................................................

120

PERSPECTIVES AND SUMMARY Converging physiological, genetic, and biochemical studies have established the salient features of preprotein translocation across the plasma membrane of Escherichia coli. Translocation is catalyzed by two proteins, a soluble chaperone and a membrane-bound translocase. SecB , the major chaperone for export, forms a complex with preproteins. Complex formation inhibits side reactions such as aggregation and misfolding and aids preprotein binding to the membrane surface. Translocase consists of functionally linked peripheral and integral membrane protein domains. S ecA protein, the peripheral mem bra ne domain of translocase, is the primary receptor for the SecB/preprotein complex. SecA hydrolyzes ATP, promoting cycles of translocation, pre protein release, .:ljiw-dependent translocation, and rebinding of the pre protein. The membrane-embedded domain of translocase is the SecY/E pro tein. It has, as subunits, the SecY and SecE polypeptides. The SecY/E protein stabilizes and activates SecA and participates in binding it to the membrane. SecA recognizes the leader domain of preproteins, whereas both SecA and SecB recognize the mature domain. Many proteins translocate without requir ing SecB , and some proteins do not need translocase to assemble into the plasma membrane. Translocation is usually followed by endoproteolytic cleavage by leader peptidase. The availability of virtually every pure protein and cloned gene involved in the translocation process makes E. coli the premier organism for the study of translocation mechanisms.

INTRODUCTION Cells are divided into multiple compartments, each a membrane or a mem brane-bounded aqueous space with a unique set of resident proteins. Com partmentation allows metabolic reactions that would otherwise lead to futile cycles to be separated and regulated and allows metabolic control through selective routing of proteins and other molecules. Eukaryotic cells have more than 20 distinct compartments, whereas E. coli has four: cytoplasm, inner (plasma) membrane, periplasm, and outer membrane. During the past two


ENZYMOLOGY OF PROTEIN TRANSLOCATION

103

decades, the pathways of protein targeting and translocation out of the cytoplasm have been heavily studied for E. coli and for eukaryotic organelles such as the rough endoplasmic reticulum, mitochondria, chloroplast, peroxi some, and nucleus. In each case, the basic questions are the same: What are the sequence elements or structural features in a protein that specify its correct target membrane for translocation? What proteins aid in protein targeting to the membrane? Is translocation via a proteinaceous transport system or through the lipid phase? What are the energetics of this process? E. coli offers unique advantages for the study of these questions. For physiological studies, cells can be grown in a defined minimal medium and pulse-labeled with radioactive amino acids. Translocation across the plasma membrane lexposes newly made proteins to externally added protease. These technical advantages have allowed extensive in vivo studies of translocation in this organism, whereas there are fewer studies of in vivo translocation into endoplasmic reticulum or mitochondria. The sophisticated genetics of E. coli has allowed the selection of mutants in the genes for both exported proteins and the pfOlteins that facilitate export ( 1) and studies of their interactions (2). Biochemical studies of in vitro translocation reactions (3-7) have allowed dissection to yield pure components, first for the precursor of M13 coat protein (8) and, more recently, for proteins with more complex translocation requirements (9-13). A rough chronology of these studies is presented in Table 1. In this review, however, we emphasize our current view of the enzymology of preprotein translocation across the plasma membrane (Figure 1) rather than chronology. An excellent review of the genetics of bacterial export has appeared recently (1). We do not review here the export of colicins and toxins by "dedicated" transport systems.

LEADER PEPTIDES Periplasmic and outer membrane proteins, as well as some plasma membrane proteins, are: made with amino-terminal leader (signal) sequences ( 14-21) that are removed immediately after translocation. Leader sequences are 16-26 residues long, and comprise a basic amino-terminal region, a central apolar region, and a nonhelical carboxy-terminal domain. Genetic studies have clearly established that the basic character of the amino terminus (22-25) and the apolar composition of the central domain (26-33) are essential for translocation. The conserved features of the last part of the leader, a helix disrupting glycyl or prolyl residue and small side-chain residues at positions -1 and -3 with respect to the cleavage site ( 14, 19), are not needed for translocation (34, 35) but are essential for recognition by leader peptidase (or, for lipoproteins, lipoprotein signal peptidase) (34, 36, 37). Exchange of leader sequences between E. coli proteins usually allows continued export


104 Table 1

A chronology of some important findings in bacterial protein export"


Year

Finding

References

1969

Sequence of the first integral membrane protein

177

1976

Fusion proteins used to study secretion

178

1977

Bacterial membrane-bound polysomes

179, 180, 193, 194

1977

In vivo kinetics of protein export

51

1978

Discovery of E. coli leader peptidase

181

1979

Loop model of bacterial secretion

168

1979 -1983

Translocation is not coupled to translation

52-56, 182, 183,195

1980

Leader sequence mutations block secretion

26,184

1980

Discovery of lipoprotein signal peptidase

185

1980

Membrane potential is needed for protein export

98-101 196

1981

Isolation of secA mutants

1981

Isolation of prLA mutants

46

1982

Isolation of pure precursor proteins

155, 156,186

1982-1983

Isulatiun of leader peptidase, lipoprotein signal

187-191,197, 198

peptidase,and their genes 1983

Isolation of SeeB mutants

48

1983

Isolation of SeeY mutants

86

Reconstitution of procoat translocation with pure com-

8

1983

ponents 1984

In terfe rence

1984

In vitro Sec-dependent translucation

5-7

1984 -1986

Preproteins are unfolded for export

164, 166 105

80

1985

ATP is needed for translocation

1985

MI3 procoat is Sec-independent

93

1988

Interference is via SecB; SecB as ch apero n e

10,49,57,64

1988

Isolation of SecA protein

II, 12

1988

Isolation of Sec B protein

9,10

1988

Isolation of SecE mutants

88, 89

1989

Suppressor directed inactivation

192

1989

SecA is the catalytic element of translocation ATPase

76

1989

Translocation intermediates

114, 168a

1990

Purification of SecY/E protein

13

1990

SDI demonstration of SecY and SecE interaction

2

"The authors offer their sincere apologies for inadvertant omissions that undouhtedly occur here.

(38, 39), albeit at a reduced rate, thereby suggesting that the leader peptide and the rest of the protein have distinct functions in export. Two kinds of genetic studies have established that these are the only features of functional leader peptides that are essential. In one study, the apolar domain of a leader was replaced by leucyl residues (29), Normal export ensued, establishing that apolarity is the only important feature of the center of leader peptides. In a similar vein, the addition of several lysyl residues at the amino terminus of an apolar membrane-spanning region can convert it into



105

a functional leader (40). In a second approach to leader function, secretion of pre-invertase was measured in yeast after the normal leader region was completely deleted and replaced by random peptides (41). Approximately one in five random peptides supported translocation. Sequence analysis showed that the onIy common theme among these peptides was simply their basic and apolar character. How can such a simple, and common, motif serve to direct the selective and efficient process of secretion? Recent studies, reviewed below, suggest that the mature domains of exported proteins also bear some of the information specifying export. In addition, there may be a selective pressure on proteins that remain in the cytosol to avoid leader-like sequences near their amino terminus (41 ). Nevertheless, the relative roles in leader function of either protein recognition or of physical partitioning into mem brane lipids remains an area of active inquiry. Leader peptides serve a number of distinct functions. The leader regions of pre-MBP (42) and pre-,B-Iactamase (43) retard the folding of the mature domain, and this delayed folding may be crucial for interaction with the SecB chaperone (see below). The leader region is directly recognized by SecA (44, 45), SecY (46), and leader peptidase (47). Direct evidence for its interaction with lipid during Sec-dependent translocation is not yet available. SEC PROTEIN-DEPENDENT TRANSLOCATION

Genetic and biochemical studies of bacterial secretion have converged, pro viding a model (Figure lA) of how proteins are selected for translocation. Export of many precursor proteins involves interaction with a cytosolic chaperone, most commonly SecB (48, 49). Most proteins (50) then use a complex, membrane-bound translocase. Periplasmic and outer membrane preproteins must be cleaved by leader peptidase to leave the outer surface of the inner membrane (35). SecB

Preprotein s ynthesis and membrane transit are not coupled in E. coli. (51-56). During the interval between synthesis and translocation, some precursor proteins are stabilized by forming a stoichiometric complex with SecB (5761), a soluble oligomeric "chaperone" protein. Though SecB is not essential for either in vivo (61 a) or in vitro (62) translocation, it prevents nonproductive reactions such as tight protein folding (63, 64), aggregation (58), or mis association with low-affinity sites on the membrane (65). It has been postulat ed that chaperones such as SecB might function by association with apolar regions (66), though there is no direct evidence. In any case, SecB clearly associates with the mature domain of precursor proteins:

106



A

B

Dependent Protein Export

Sec

Catalytic

Cycle

of Preproteln

Translocation

CYTOPLASM Figure 1 Sec-dependent protein translocation. Modified from A. Hartl et al (65) and B. Schiebel et al (84). Small case numbers in part A refer to the binding of 1 . SecB to the mature domain of a precursor pro te in , 2. SeeB to SecA, 3. SecA to the leader domain, 4. SecA to the mature domain of a precursor protein. and 5, SecA to SecY/E.


ENZYMOLOGY OF PROTEIN TRANSLOCAnON

107

1. SecB can bind to mature domains of precursor proteins in vitro (57, 63, 67). 2. Exchange of leader regions between pre-maltose-binding protein, which interacts strongly with SecB, and pre-alkaline phosphatase or pre-ribose binding protein, which do not, has shown that SecB-dependence is con ferred by the mature domain, not the leader (60; D. Collier, S. Strobel, P. J. Bassford Jr., personal communication). 3. Overproduction of a mutant pre-maltose-binding protein that lacks its leader region interferes with the export of wild-type precursor proteins (68, 69). This interference is overcome by overproduction of SecB pro tein, thus indicating that the interfering maltose-binding protein was titrat ing the available SecB protein. 4. Regions of interaction with SecB have been genetically defined within mature domains of precursor proteins (E. Altmann and S. Emr, personal communication). Though SecB associates with the mature domain of preproteins, the leader region, by retarding the folding of the mature domain, may strongly influence binding to SecB (64).

There is no evidence that SecB functions as an "unfoldase." Association with SecB prevents pre-maltose-binding protein from folding into its final, stable folded structure (64), which cannot cross the membrane. This does not show, however, that the preprotein is being held in an unfolded state, devoid of stable secondary or tertiary structure, or being actively unfolded once folding occurs. ProOmpA in complex with SecB has been shown to have substantial folded structure (58). In this case, the SecB association shields apolar surfaces of proOmpA and prevents its aggregation. SecB is the major chaperone for preprotein export, yet other chaperone proteins have a role as well. GroEL selectively facilitates the secretion of pre-f3-1actamase (70) and DnaK can support the secretion of artificial fusions between leader peptides and f3-galactosidase (71). Either GroEL or trigger factor, an abundant cytosolic protein, can stabilize proOmpA for in vitro translocation across membrane vesicles (57). Genetic studies show that the interaction of proOmpA and trigger factor occurs in vivo, but is actually inhibitory for export (72). The biochemical basis for the unique role of SecB, rather than the more abundant GroEL (73) or trigger factor (72), may lie in the specific affinity of SecA for SecB (65). SecB may contribute a major share of the binding affinity of the SecB/preprotein complex for SecA (65). SecB can even stabilize proOmpA while they are both bound to the membrane via SecA (65). Several salient questions remain about the mechanism of SecB function. How is a I: 1 binding stoichiometry achieved by an oligomeric protein? Perhaps the several SecB subunits associate with several different apolar

108


domains of the bound preprotein. What features of the mature domains of precursor proteins are recognized by SecB? Crystals of a SecB/preprotein complex may be essential for clear answers to these questions. Finally, the

nanomolar binding affinity of SecB for SecA suggests that it might remain bound to SecA in the high protein concentration of the cell. In this light, even SecB could be viewed as a subunit of the peripheral domain of translocase.


SecA

SecA is a peripheral membrane protein (12, 74) of 102 kDa (75). It serves as the high-affinity receptor for the SecB/preprotein complex (65). SecA hydro lyzes ATP and couples this energy to translocation (76, 77). SecA function and high-affinity membrane binding require acidic lipids (78; J. Hendrick, W. Wickner, in preparation) and the membrane-embedded SecY/E protein (65). The synergy of action of suppressor mutants in SeeA(prlD) (45) and SeeY (prIA) (79) is consistent with the physical interaction of these proteins. SecA and SecB are apparently the sole peripheral membrane proteins with direct roles in translocation (12, 13). The binding of SecA to the membrane strongly enhances its affinity for the SecB/preprotein complex (65). SecA binds this complex by virtue of its affinities for SecB (65) and for both the leader (44, 80) and mature domains of the precursor protein (78). Basic residues of the leader are especially important for its recognition by SeeA (8 1 , 82). SecA requires both SecY/E protein and acidic lipids for high-affinity binding to the membrane. This requirement for acidic lipids may be one reason for the requirement for these lipids in the overall translocation process (83). When SecA protein is in contact with each of the essential elements for transloca tion-a preprotein with an authentic leader and mature domain, the SecY/E protein, and acidic phospholipids (78)-it hydrolyzes ATP. This activated ATPase activity is termed the "translocation ATPase," since it depends on each of the elements needed for successful translocation (76). ATP binding to SecA drives a limited preprotein translocation (84). Model reactions suggest that ATP hydrolysis drives a release of precursor from SecA (84). Llfiw driven translocation occurs when the preprotein is not bound to SecA nucleo tide. SecA, ATP, Llfiw, and SecY/E protein are required for translocation as the protein progresses across the membrane (84). Each preprotein undergoes many cycles of SecA binding, ATP binding to SecA, limited preprotein translocation, ATP hydrolysis, release, Llfiw-driven translocation, and rebinding (84). The progress of the translocation reaction is readily reversible (84). The two sources of metabolic energy for translocation, ATP and Llfiw, govern progress across the membrane at each stage (Figure lB). SecA is clearly a complex enzyme with multiple catalytic and regulatory interactions. Its synthesis is regulated by the overall functionality of translo case (85), underscoring its central role in selecting proteins for export. Its



1 09

amino acid sequence has three potential nucleoside-triphosphate-binding motifs (D. Oliver, personal communication), and the protein can be covalent ly modified by up to three moles of N3-ATP per mole of SecA (76). Hydrolytic and regulatory roles have not yet been assigned to the individual ATP-binding sites. ATP hydrolysis is regulated by each of the essential components of translocation-SecY/E protein, the preprotein itself, and acid ic lipids. Even competitive inhibition of translocation ATPase by synthetic leader peptide is manifest as an increase in the Km for ATP (44). In tum, ATP hydrolysis promotes preprotein release (84). Upon interaction with lipids, SecA is rendered thermolabile, but is stabilized by its interactions with ATP, SecY/E, and the leader and mature domains of precursor proteins (78). Strikingly, the stabilization of the ATPase activity of SecA by precursor protein can be efficiently mimicked by a mixture of synthetic leader peptide and the mature protein (78), affording enzymologists an opportunity to ex plore directly the separable specificities of SecA for the leader and mature domains of preproteins. SecYIE

Both biochemical ( 1 3) and genetic (2) studies have established that the SecY/E protein is the integral membrane protein that supports translocation. It constitutes approximately 0.6% of the protein of the plasma membrane, and comprises three subunits, the SecY polypeptide (86, 87), the SecE polypeptide (88-90), and a polypeptide of unknown gene. The latter subunit, termed band 1 , copurifies with the SecY and SecE subunits during either column chromatography or immunoprecipitation (13). Confirmation of its role in translocase, however, will have to await either determination of its role in the mechanism of translocation or the identification and mutation of its gene. Although the transbilayer topology of the SecY and SecE polypeptides is known from genetic studies (90, 9 1 ), the number and stoichiometry of each subunit in the SecY/E protein is not yet known. SecY and SecE are ex ceptionally hydrophobic proteins; the former can even aggregate in the pres ence of SDS (87) and is not solubilized from the plasma membrane by most common detergents (44). In order to solubilize and maintain the SecY/E protein in 2L form that was isolable and could be reconstituted with lipid to support functional translocation, it was necessary to employ a combination of detergent and lipid mixed micelles in solutions with lowered water content, achieved by glycerol addition (92). Liposomes bearing the purified SecY/E protein support a translocation reaction that requires SecA, ATP, and an authentic preprotein and responds to an electrochemical potential (13). Equiv alent translocation units of inner membrane vesicles and proteoliposomes with purified SecY/E protein have almost equivalent amounts of SecY protein (13), a result that suggests that no other proteins are essential for the basic


1 10


translocation reaction. Since SecA is bound to the membrane at the SecY/E protein (65), we have defined "preprotein translocase" as the combination of SecY/E protein with its bound SecA. The former constitutes an integral membrane domain of translocase, whereas the latter is its peripheral domain. There are more questions than answers about the basic functions of the SecY/E protein (92a). Is the energy of ATP hydrolysis transferred between SecY/E and SecA, much as energy is transferred between the integral and peripheral domains of the FJFo-ATP synthase? The availability of pure SecYI E may allow its assay as a proton-conducting channel, perhaps gated by the preprotein. Does the SecY/E protein serve as a transport system for the preprotein, conducting it through its center or along its surface? The existence in this gene of prlA mutants (prlA seeY), which are strong suppressors of leader sequence mutations (46), indicates that the SecY/E protein interacts directly with the leader peptide region during translocation. This function is now open to direct biochemical test in the reconstituted translocation reaction. =

TRANSLOCATION THAT IS SEC-INDEPENDENT Though conditionally lethal mutants in the see genes affect the secretion of most bacterial proteins, certain small proteins such as M 1 3 procoat protein (93), PF3 coat protein (A. Kuhn, personal communication), and honeybee prepromelittin (94) do not require the Sec proteins for translocation. This c annot simply be a question of size, as secretion of the 8100 Mr precursor of heat-stable enterotoxin (prepro-STB) requires SecA (95) and the MalF protein requires neither SecA nor SecY for its membrane insertion (I. McGovern, B. Traxler, J. Beckwith, personal communication). M 1 3 procoat protein, the first preprotein found not to require Sec proteins for membrane insertion (93) (Figure 2), has a typical leader sequence (96). Genetic exchangc of thc leader domains betwecn M13 procoat and proOmpA, a Sec-dependent protein, showed that Sec-dependence was a feature of the mature domain rather than a particular property of the leader region (39). This was also established by point mutations, which showed that each part of the mature domain of the protein is essential for its assembly into the membrane (31, 32). A similar conclusion has been reached for prepromellitin insertion into the endoplasmic reticulum (97). This is in striking contrast to the Sec-dependent preproteins, in which few point mutations in the mature domain are reported to have a severe effect on export. What is the relationship between the Sec-dependent and Sec-independent pathways of protein translocation? Occam's razor suggests that features of translocation shared between these pathways reflect a shared underlying mechanism. SeC-dependent and Sec-independent proteins have typical N terminal leader sequences, thereby suggesting a role in all translocation for


PER/PLASM

�

�

5

:\.

�� C

lYe Cleavage by N

AfY-.£-C

CYTOPLASN

__

Leader Pe pt idase

a --< o "rl

'"0

� �

;l >

z v:> t""' o n Figure 2

The Sec-independent insertion of M 1 3 procoat into the plasma membrane of E. coli. From Kuhn et al (32).

� o z

-

1 12


the leader region that goes beyond its recognition by SecA. Since Sec independent translocation requires the membrane electrochemical potential, the potential may also act on the Sec-dependent preproteins per se rather than solely via a postulated proton/protein antiport mechanism of SecY/E.

ENERGETICS OF EXPORT


Translocation requires two forms of energy, ATP, and .ditw. In vivo dissipa tion of .dAw by iono- or protonophores arrests translocation, and precursor

proteins accumulate within the cell (54, 55, 98- 104). Evidence that both ATP (105) and .ditH+ ( 106, 107) are involved in translocation emerged from in vitro studies. The requirement for ATP is absolute, whereas .ditw is strongly stimulatory.

The Role of LlflH+ The requirement for .1itH+ varies, depending on the precursor protein species (l08). Observations that .dJtH+ is only stimulatory in vitro, whereas it is clearly required in vivo, led to the suggestion that the role of .1Aw is indirect

( 10 5). Compelling evidence that .1,uw is an intrinsic part of the translocation mechanism was provided by studies with the reconstituted translocation reaction ( 1 3, 84, 109) .1,uH+ stimulates translocation in a reaction with purified components that utilizes both the transmembrane electrical potential .11/1 and the transmembrane pH gradient .1pH ( 109), as it does in vivo (104). .1pH may perrorm a dual function in translocation ( l09), acting as a direct energy source and indirectly modulating the rate of translocation via the effect of the cytosolic pH on the activity of the translocase . .dAw may directly affect the precursor protein, activate SecY/E to promote transport, or both. Insofar as the .1Jtw acts directly on precursor proteins during translocation, the roles of .1pH and .11/1 may be different. .1pH may drive translocation by promoting the facile deprotonation of positively charged residues prior to membrane transit and their protonation afterward ( 10 9). .11/1 may have an electrophoretic effcct to promote the transfer of regions bearing a net negative charge. Interactions of the N-terminal domain of the leader sequence with the surface of the membrane may be followed by an electrophoretic transmembrane alignment of the internal dipole of the leader in response to .dl/l (100). Charged residues provide a barrier to translocation, as illustrated by the strict "positive inside-rule" that guides the membrane topology of polytopic membrane proteins (40, 1 10, I l l ). Positive ly charged residues appear to be significantly more difficult to translocate than negatively charged ones ( 1 12). The .1itw could serve directly as a driving force for translocation if polypeptide transit requires concurrent proton move.


113

ment in the opposite direction. Proton transfer by a proton/polypeptide anti port mechanism could be gated in response to the distribution of positive charges in the precursor protein or obey a strict coupling stoichiometry. The SecY/E protein may conduct such proton movements, a biochemical function that can be directly tested with the reconstituted translocation reaction.


The Role of ATP ATP promotes translocation solely via its hydrolysis by the SecA protein (76). SecA may use the energy of ATP binding to translocate small domains of the precursor (84), whereas the energy of ATP hydrolysis drives preprotein release (84). LljLw appears to have a role very different from ATP hydrolysis (Figure lB). Although SecA may allow LljLw-independent translocation ( 113), LltLH+ has no significant effect on the kinetics of SecA binding and function ( 1 09). Early and late stages of proOmpA translocation are reported to differ in their requirement for ATP and Ll,uw (84, 109, 114, 115). The very first stage, a limited translocation that allows processing of the leader peptide, strictly requires ATP (84). Subsequent translocation can be efficiently driven by either SI�cA and ATP or by LljLw alone (84). Studies of the kinetic isotope solvent efkct show that the overall rate of translocation is limited by a critical proton transfer reaction (109). This involvement of catalytic protons provides further support for a direct role of LljLH+ in translocation. COMPARISONS WITH OTHER PROTEIN TRANSLOCATION REACTIONS

A number of eukaryotic membranes are competent for protein translocation, including the membranes of the endoplasmic reticulum (ER) ( 1 16), mitochon dria (117, 118), chloroplasts (19), and peroxisomes (120). All protein transport appears to follow certain common principles: 1 . Proteins to be transported contain contiguous targeting sequences. 2. The membranes expose receptors or receptor-like components at their cytosolie surfaces. 3. Except in special cases, translocation requires enzymic catalysis and metabolic energy in the form of ATP or GTP and, for some, a membrane potential. 4. Preproteins have to be in a translocation-competent conformation, prob ably loosely folded, which is maintained by their interaction with molecu lar chaperones. Comparison of different translocation reactions (Table 2) suggests general mechanisms of protein translocation and highlights important unresolved questions.

Table 2

A comparison of bacterial protein export with protein transport into endoplasmic reticulum and

mitochondria Bacterial protein export Targeting signals

1 6 -26 residues; positively charged N-terminus followed by central apolar region .

Cytosolic chaperones

SecB, GroEL, Hsp70 (Dnak).

Recognition

Via peripheral membrane components SecB and SecA. Recognition of SecBI

Hydrophobic moment along the long axis of the polypeptide chain.

preprotein complex through affinity of SecA for SecB and for leader and mature parts of the preprotein. Subsequent interaction with phospholipid or SecY/E.


Sequential receptor cascade. Bypass of SecA by M l3 procoat. Translocation

Across single membrane; requires integral membrane protein SecY/E.

Proteolytic processing

Cleavage by membrane integrated enzymes leader peptidase or lipoprotein signal

Energy requirement

Cytosolic ATP (SecA-ATPase) and LlMH+ required th rou gho u t translocation. Slow

peptidase.

membrane transit possible without LlMH+, and small parts of polypeptide chain can translocate independently of ATP. Transport across ER membrane Targeting signals

As for bacterial protein export, ER and bacterial signal sequences are functionally


Hsp70 (SSA) in posttranslational transport in yeast; may be functionally replaced

Recognition

b y tightly coupled cotranslational tran,location in mammalian cells. Via SRP and docking protein in mammalian cells. Sequential receptor model .

Translocation

Across single membrane. Requites "signal sequence receptor" (SSR) in mamma

interchangable.

Bypass o f SRP/docking protein b y small precursors. lian cells and integral membrane proteins Sec61-63 in yeast. Sequence homolo gy between Sec6 1 and bacterial SecY . Proteo] ytic processing

Cleavage b y membrane-integrated signal peptidase. Cleavage specificity similar to

Energy requirement

GTP required for SRP/docking protein. ATP-requirement for release of hsp70

bacterial leader peptidase. (SSA) in yeast. ATP in the ER lumen for function of hsp70 (BiP) in transloca tion and folding/assembly of proteins.

Mitochondrial protein import Targeting signals

1 0 -70 residues; rich in po�itively charged and hydroxylated residues . Can form amphiphilic a-helices. Hydrophobic moment perpendicular to the long axis of the polypeptide chain.


Hsp70 (SSA).

Recognition

Via multiple surface receptors in the outer membrane followed by outer membrane general insertion protein. Converging receptor model. Bypass of surface recep tors possible.

Translocation

Across two apposed membranes at translocation contact sites. Requires hsp70

Proteolytic processing

Presequences completely translocated into matrix space. Cleavage by metal

Energy requirement

Cytosolic ATP for release of hsp70 (SSA). ATP in the matrix for function of

(SSC!) in the matrix. dependent, soluble enzyme. hsp70 (SSel ) and hsp60. AI/! across inner membrane to trigger insertion or

translocation of positively charged leader. Membrane transit of mature domain is possible independent of Lll/!.


1 15


Targeting and Recognition The signals required to direct noncytosolic proteins to their target com partments are mostly located at the amino terminus as cleavable presequences; however, proteins having internal targeting sequences are known. In general, the proteolytic cleavage of presequences, performed by specialized proteases ( 1 2 1 - 1 23), is not necessary for translocation. There is no sequence similarity among the targeting signals for each compartment. Hence the relevant in formation must reside in more degenerate features, such as the distribution of charges, hydrophilicity and hydrophobicity, and secondary structure. Bacterial and ER signals are almost indistinguishable and are functionally interchange:able ( 1 24-1 26). In contrast, the typical mitochondrial matrix targeting sequence lacks contiguous hydrophobic segments (127) but has several positively charged and hydroxylated residues. These sequences can form amphiphilic a-helices, with basic residues exposed on one face and uncharged and hydrophobic residues on the other (16, 17, 128). Amphiphilic ity is also �l structural feature of all bacterial and ER targeting signals due to the linear arrangement of polar and apolar regions. Thc structural differences between secretory and mitochondrial targeting signals probably correspond to mechanistic: differences of the initial steps of translocation. Targeting and recognition involve the interaction of preproteins with cytosolic pmteins as well as with peripheral and integral membrane com ponents. In eukaryotes such as yeast, cytosolic heat-shock proteins of the hsp70 family may interact with secretory and mitochondrial preproteins, stabilizing them in translocation-competent conformations ( 1 29, 1 30). Chaperone-preprotein interactions are also a common theme in the bacterial cytosol. However, the function of the main prokaryotic secretion chaperone, SecB, is dilitinguished by its specific interaction with the receptor SecA, an interaction that contributes to the targeting of the preprotein-SecB complex (65). In mammalian cells, preproteins interact via their leader sequences with the signal rl�cognition particle (SRP) ( 1 3 1 ) as they emerge from ribosomes. This may slow translation until the complex of ribosome, SRP, and nascent chain has bound to the SRP receptor ( 1 32), or docking protein ( 1 33), at the ER membrane, thus ensuring cotranslational translocation. However, several studies ( 1 34-137) have shown that large domains of preprotcins can be made prior to initiating successful translocation. SecB and SecA appear to exert part of the functions ascribed to SRP without being structurally related to it. SecA recognizes SecB (65) as well as the leader and mature parts of the preprotein and SecB stabilizes the mature part of the precursor. SRP from dog pancreas can substitute for the stabilizing function of SecB in bacterial protein export in vitro (62). llhe complex of preprotein and SecB "docks" at its receptor, the "translocase," comprising SecA and membrane- embedded SecY/E protein (65).

116


Nuclear-encoded mitochondrial proteins encounter their target compart


ment by directly binding to receptor proteins integrated into the mitochondrial

outer membrane (118). An interaction with cytosolic components specific for the targeting sequences seems not to be required. Receptor components have been identified (138-140) that have overlapping specificity. The structural elements in precursor proteins recognized by these receptors are unclear. Receptor-bound precursors converge to a common membrane insertion site, the general insertion protein GIP (141). This component is probably located at contact sites in which outer and inner mitochondrial membranes are closely apposed.

Translocation and Energy Requirement Investigators of ER and mitochondrial protein translocation favor the idea of a proteinaceous pore or channel that preproteins are thought to penetrate in extended conformations ( 142-144). In the case of mitochondria, such a transport protein could span the two membranes at contact sites ( 145, 146). Alternatively, it might only span the inner membrane and be connected to the apparatus for binding and insertion in the outer membrane. Candidates for the ER translocase are the membrane-integrated products of the SEC61-63 genes in yeast (147) and the 35-kDa signal sequence receptor (SSR) in mammalian ER ( 148). Preproteins may reach the SSR either directly or via SRP and docking protein. The SRP and docking-protein-independent pathway seems to be followed by small precursor proteins. Interestingly, one of these proteins, prepromellitin, can also be exported across the plasma membrane in E. coli in the absence of SecA and SecY function (94). The Sec61 protein has homolo gy to SecY in cytosolic and membrane-embedded parts (R. Schekman, personal communication), again underscoring the similarity between the bacterial and ER systems. Bacterial and mitochondrial protein transport require both ATP and the inner membrane electrochemical potential. The requirement for cytosolic ATP is connected to keeping the preprotein in a more open conformation or perhaps even actively unfolding it during translocation. SecA may be such an ATP-driven unfoldase. In the case of mitochondria, cytosolic ATP is prob ably required for the release of hsp70 from the receptor-bound precursor ( 118, 149). In striking contrast to the bacterial system, both ER and mitochondria have an additional requirement for ATP on the trans-side of the membrane ( 150-152). The energy input by SecA and ATP on the cis-side of the bacterial membrane could replace the functions performed by components that bind to the translocating chain on the trans-side in other translocation reactions. As long as the part of the bacterial protein that has not yet translocated is actively unfolded, translocation may be driven by the energy derived both from L1{Lw



117

and from folding in the periplasm. Movement of the polypeptide across the membrane is readily reversible, and ATP-hydrolysis and/or .1,uw is required to achieve unidirectionality (84). In contrast, reversibility of chain movement has not been observed in mitochondria, perhaps because of the presence of efficient binding proteins on the trans-side of the membrane. An additional possibility is that SecD and SecF of the E. coli inner membrane support translocation by binding to translocating preproteins as they emerge into the periplasm ( 153). The mechanism of such an interaction, however, would have to differ from that suggested for hsp70 proteins in the lumen of ER and mitochondria, since ATP is absent from the bacterial periplasm. The proton-motive force accelerates each stage of the movement of pre proteins across the bacterial plasma membrane (84, 109). In contrast, the mitochondrial system utilizes the electrical component .11/1 only to drive the translocation of the positively charged leader sequence across the inner membrane {l54), perhaps by a direct electrophoretic effect of 111/1 (negative inside). Notably, bacterial proteins move across the membrane in the opposite direction with respect to the polarity of the membrane potential as in mitochondria. However, the basic N-terminus of the bacterial leader apparently remains on the cytosolic side of the membrane, whereas the basic residues of the mitochondrial leader cross into the matrix.

CURRENT QUESTIONS IN BACTERIAL EXPORT Procoat

How does M13 procoat translocate without Sec-protein-mediated catalysis? If the small size of M13 procoat inherently prevents its stable folding, it would be unnecessary for SecA to catalyze unfolding. As one proposed function of SecB is to shield apolar surfaces, the two apolar regions of procoat may be apposed to each other prior to membrane interactions. The three charged segments of procoat may also function to shield these apolar domains; indeed, the character of each of these segments is important for procoat membrane insertion. However, our enthusiasm for these ideas is tempered by the low solubility of purified procoat (155, 156). A second question is what blocks procoat recognition by SecB and translocase when a mutation is introduced into the mature domain of procoat that prevents its Sec-independent transloca tion? The answer to this question may lie in the recognition specificity of SecB and SecA for the mature domains of exported proteins. Mature Domains

Initial studies of protein translocation focused on the leader sequence; howev er, simple joining of an authentic leader to a cytosolic protein does not always


118


guarantee its rapid and efficient export (157). Large deletions in the mature region of an exported protein can dramatically lower both the rate and extent of export (158). One apparent constraint on the mature sequences of exported proteins is that lysyl and arginyl residues are excluded from the first few residues after the leader (16, 17, 50, Ill , 159, 160). It is not known why these basic residues would interfere with translocation. Acidic amino acids often occur 3-4 residues (one tum of an a-helix) from basic residues in exported proteins (161), suggesting a role for charge pairing in translocation. Another possible constraint on mature regions is that they must not fold in a manner that obscures the leader from recognition by SecA or that prevents unfolding. This would lead to the testable proposal that cytosolic proteins, given a leader sequence, would bc more readily exported if rendered structur ally labile by genetic means. Finally, SecB and/or SecA may recognize features of preprotein charge, polarity, or secondary structure. In any case, the existence of a stable ternary complex among SecB, a preprotein, and membrane-bound SecA (65) suggests that SecB and SecA recognize different aspects of preproteins.

Unfolding Only a few studies have been made of the state of preprotein folding. Studies of protease sensitivity (162, 163) show that proteins emerge into the peri plasm in an initially unfolded state. Prior to translocation, leader peptidase is far more susceptible to digestion in cell lysates than after it has assumed its final trans-membrane form (164). Pre-MBP must remain unfolded to some degree in order to translocate (165, 166), though the degree of secondary or tertiary structure in the export-competent preMBP is not yet known. Trimethoprim, by stabilizing the folded form of a proOmpAldihydrofolate reductase fusion protein, prevents its translocation (167). ProOmpA, in com plex with SecB, has significant secondary and tertiary structure (58). If, as is widely assumed, exported proteins cross the membrane as a simple loop (39, 168), an active unfolding process would be necessary. Although there is no direct evidence, it is possible that SecA could actively unfold preprotcins at the expense of ATP hydrolysis.

Translocation Pathway Do preproteins cross the membrane through a SecY/E "pore," through the lipid phase, or along a special surface of the SecY/E protein? It has recently been shown that proOmpA can still translocate when its cysteines are joined as a disulfide, forming a protein loop ( l68a), or when the cysteines are derivatized by coumarin (58) or N-ethylmaleimide (84). Since the plasma



119

membrane remains i mpermeant t o protons at all times, i t could not have a water-filled pore such as is found in the outer membrane. It seems unlikely to us that a transport protein for preproteins could have the structural flexibility to accommodate a loop or a coumarin and yet remain "proton-tight." The establishment of conditions for accumulating preproteins as translocation intermediates (84, 1 1 4) may now allow assessment of their association with SecY/E or accessibility to lipid-embedded probes. The requirements for SecA, ATP hydrolysis, and L1jLw persist throughout the course of transloca tion (84, 1ll4, 1 15), demonstrating that the role of SecA goes beyond simply serving as a receptor.

Role of Lipid Although we have emphasized the role of the translocase enzyme in protein export from E. coli, the lipid phase also plays a vital , if poorly understood, role. Acidi,� lipids (78 , 83; J. Hendrick, W. Wickner, in preparation) are required for high-affinity SecA binding to the membrane and for the activa tion of translocation ATPase. Intriguing reports have suggested that features of the fatty acyl phase, such as fluidity, may play a major role in the translocation process (51 , 1 69). A large body of data shows that leader peptides mUtst retain their capacity to partition into lipid to be functional ( 1 70, 1 7 1 ). Whether or not they do so during the translocation process remains to be determined.

Role of JJ iiH+ Some of thf: possible roles of L1jLH+ in translocation are outlined above. The reconstitution of translocation with all purified components ( 1 3) may allow a more quantitative study of the effects of L1jLw.

Folding in the Perip lasm Newly made proteins must be cleaved by leader peptidase for release into the periplasm (35), and may reside on the outer surface of the plasma membrane for a brief period after membrane transit ( 1 60). In other translocation reac tions, Hsp60 catalyzes protein folding after import into mitochondria (151 , 1 72) and chloroplasts ( 1 7 3). BiP may assist the folding of proteins as they enter the ER (RER) and appears to be required for translocation ( 1 5 0, 1 74). SecD and SecP, which are largely exposed to the periplasm, might catalyze periplasmic protein folding ( 1 75). The rapid rate of protein export in vivo might require SecD and SecP action to "clear" the periplasmic surface of the SecY/E protein for further rounds of translocation. The slower rates and lower extents of in vitro translocation may render detection of the SeeD and SeeP catalysis difficult.

l20



Role of Translation Translocation in E coli is clearly not coupled to ongoing polypeptide chain growth. Nevertheless, it is premature to conclude that no distinctions exist between polypeptides that happen to initiate translocation while still attached to the ribosome and those that cross entirely posttranslationally. A major aspect of SecB and SecA function relates to the folded state of the protein, as noted above. The polypeptides that cross cotranslationally may not fold prior to translocation and therefore might be less dependent on possible SecA "unfolding" or SecB chaperone functions. Thus, in SecA- or SecB-deficient strains, the export defect is dramatically relieved by drugs or mutants that selectively slow protein synthesis ( 1 65, 1 76), and the dependence of translocation on SecA or SecB is diminished when only a small domain of the polypeptide, which might not fold as stably, remains to be translocated (84, 1 1 4, 1 1 5).

Regulation The processes of secretion and membrane assembly must now be dovetailed with other elements of what has been called "Project K I2," the attempt to understand the interrelationships of the essential systems of one of the sim plest free-living organisms. Export can be regulated by the rates of transla tion, by the levels of ATP and Ll,uw, and by the synthesis of SecA. It will, in turn, be interesting to know how the cell measures when sufficient cell surface growth has occurred to warrant ccll division or another round of replication.

PROSPECTUS Our current limited understanding of bacterial export provides tantalizing glimpses of how nature has solved some basic problems. Specificity may be achieved by a cascade of receptors, SecB, SecA, and perhaps SecY/E, acting in series to "proofread" the choice of proteins. Leader and mature interactions may prevent final, stable folding that would block export, and the metastable, "export-competent" folded state can be supported by a chaperone. Transloca tion itself is driven by two distinct energy sources, acting in distinct manners. These ideas, and others, are now accessible to more rigorous biochemical and genetic tests. The facile interplay between genetics and biochemistry in E. coli should allow researchers during the coming decade to obtain a clear view of the mechanisms of bacterial protein export. This knowledge will enrich other areas of inquiry, such as the fundamental problem of protein folding. Progress in understanding the bacterial export reaction may also help to accelerate the parallel studies in eukaryotic and mammalian systems.


12 1


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10 2.

103. 104. 105.


1 06.

1 07 .

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SecA protein: autoregulated initiator of secretory precursor protein translocation across the E. coli plasma membrane.

[Translocation mechanism of presecretory protein across the cytoplasmic membrane of Escherichia coli].

Translocation of a folded protein across the outer membrane in Escherichia coli.

A protein with sequence identity to Skp (FirA) supports protein translocation into plasma membrane vesicles of Escherichia coli.

Correct insertion of a simple eukaryotic plasma-membrane protein into the cytoplasmic membrane of Escherichia coli.

Inhibitors of Protein Translocation Across the ER Membrane.

SecY and integral membrane components of the Escherichia coli protein translocation system.

An ATP-binding membrane protein is required for protein translocation across the endoplasmic reticulum membrane.

A 30-residue-long "export initiation domain" adjacent to the signal sequence is critical for protein translocation across the inner membrane of Escherichia coli.

Genetics and enzymology of DNA replication in Escherichia coli.

ATP11C Facilitates Phospholipid Translocation across the Plasma Membrane of All Leukocytes.

Protein targeting to and translocation across the membrane of the endoplasmic reticulum.

The stable BRP signal peptide causes lethality but is unable to provoke the translocation of cloacin DF13 across the cytoplasmic membrane of Escherichia coli.

Mechanism of glutamate-aspartate translocation across the mitochondrial inner membrane.

Precursor protein translocation by the Escherichia coli translocase is directed by the protonmotive force.

Solubilization and functional reconstitution of the protein-translocation enzymes of Escherichia coli.

Proton translocation and the respiratory nitrate reductase of Escherichia coli.

Measuring the Viscosity of the Escherichia coli Plasma Membrane Using Molecular Rotors.

Evidence for binding protein-independent substrate translocation by the methylgalactoside transport system of Escherichia coli K12.

Protein translocation across mitochondrial membranes.

Reconstruction and modeling protein translocation and compartmentalization in Escherichia coli at the genome-scale.

Organization of dimethyl sulfoxide reductase in the plasma membrane of Escherichia coli.

Oligomerization of the bacteriophage lambda S protein in the inner membrane of Escherichia coli.

Interaction between two major outer membrane proteins of Escherichia coli: the matrix protein and the lipoprotein.