Proc. Natl. Acad. Sci. USAVol. 83, pp. 5033-5037, July 1986Biochemistry

Mutations affecting the signal sequence alter synthesis andsecretion of yeast invertase

(signal peptide/recombinant DNA/protein secretion/proteolytic processing)

DANIEL PERLMAN, PATRICIA RANEY, AND HARLYN 0. HALVORSONDepartment of Biology and Rosenstiel Basic Medical Sciences Research Center, Brandeis University, Waltham, MA 02254

Communicated by Alfred Nisonoff, March 20, 1986

ABSTRACT Insertion mutations previously constructedwithin the proximal region of the yeast invertase signal se-quence did not interfere with secretion or glycosylation of theenzyme. We now describe deletion mutations within the samesignal sequence. Large deletions truncating the hydrophobiccore of the signal peptide prevented both secretion andglycosylation of the enzyme and increased the intracellularconcentration of nonglycosylated invertase. This increase wascoupled with the appearance of a new invertase polypeptide, 2kilodaltons larger than cytoplasmic invertase. The new poly-peptide was consistent in size with uncleaved (signal peptideintact) pre-secretory invertase previously identified by using invitro translation (apparent molecular mass, 62 kilodaltons).The data on enzyme activity indicate that invertase whosesecretion is aborted by large deletion mutations augments thenormal pool of cytoplasmic invertase found in sucrose-ferment-ing yeast cells.

The glycosylated secreted (S) and nonglycosylated cytoplas-mic (C) forms of invertase (EC 3.2.1.26) are encoded byseparate mRNA transcripts synthesized from a commonSUC structural gene in Saccharomyces cerevisiae (1, 2).A labile amino-terminal signal peptide utilized in the cotrans-lational secretion of preinvertase through the endoplasmicreticulum (ER) [evidenced by in vitro studies (1, 3)], distin-guishes the primary structures of the S and C polypeptides (3,4). There are two functions identified with the signal se-quence coding segment of the invertase gene. These involvethe sequence's roles as both an mRNA regulatory elementand an enzyme secretory element. The 57-nucleotide signalsequence lies within the SUC2 gene at the beginning of thecoding region for S invertase and extends to within 3nucleotides of the initiator methionine (position +61) for Cinvertase (5). The largest mRNA, which encodes the glucose-regulated S invertase, begins 40 nucleotides upstream fromthe ATG for the signal peptide, while several shorter mRNAsencoding C invertase commence within the signal sequenceitself near its distal end (4). We have previously reported thatlevels of C mRNA are regulated under a variety of physio-logical conditions, including glucose derepression and heatshock, as well as during cell cycle growth (6). Fluctuations inC mRNA levels relative to S mRNA levels are small.Nevertheless, the observation of differential as well ascoordinate changes in C mRNA levels relative to changing SmRNA levels (6) raises interesting regulatory questions.Recently we have shown that AGCT tandem DNA duplica-tions at the two HindIII sites, one just upstream of and onewithin the signal sequence coding region, significantly in-crease the mRNA and enzyme levels for both S and Cinvertase (D.P., P. Brown, P.R., and H.O.H., unpublisheddata).

We have previously shown that three very different peptideinsertions near the amino terminus of the signal peptide didnot alter invertase secretion or activity levels (8). Eachinsertion was followed by a minimum of 11 of the originalcontiguous hydrophobic amino acids. It was concluded thatthe secondary structure of the altered region (preceding thehydrophobic core) was noncritical to secretion. As for thehydrophobic core, a structural survey analysis of diversesignal peptides shows that, at a minimum, approximately 8hydrophobic amino acids capable of forming an a-helix orp8-strand structure are conserved in this element (9). VonHeijne (10) and Engelman and Steitz (11) have suggested thata sufficient free energy, AG, must be liberated in the signalpeptide-transmembrane insertion process to permit success-ful secretion. If von Heijne's estimate of the free energybarrier to secretion (85-105 kJ/mol) is correct and thepredominant signal peptide hydrophobic amino acids liberateapproximately 10 kJ/mol during partitioning (10), then 8hydrophobic amino acids may represent a functional lowerlimit for secretion to occur. Another possibility is that ana-helix or p-strand of sufficient length is required for bindingof the nascent signal peptide to the signal recognition particle(12, 13), to promote secretion. To further characterize struc-tural requirements for a functional signal peptide core, wehave generated in-frame deletion mutations in this region andanalyzed them. We have simultaneously obtained informa-tion on the regulatory role played by the invertase signalsequence coding region.

MATERIALS AND METHODSPlasmid Construction. Plasmid construction and isolation

methods, linker insertion procedures, restriction mapping,and transformation ofEscherichia coli strain JM83 have beendescribed previously (8, 14). The construction ofpAB243 andpAB202 has also been described (8). Deletions (Fig. 1) wereproduced by using BAL-31 "slow" exonuclease (Inter-national Biotechnologies, New Haven, CT). The tryptophan-requiring E. coli strain JA300 (15) was utilized to selectplasmids containing the TRPI-ARSJ yeast replicator element(16). SUC2 gene variants shown in Fig. 2 were verified byrestriction mapping and by DNA sequencing using the Sangermethod (17) modified by W. Theurkauf, H. Baum, J. Bo, andP. Wensink (personal communication). A synthetic DNAsequencing primer extending 5'--3' from position 150 to 133(see ref. 5 for nucleotide sequence) was kindly provided byK. Bostian (Brown University).

Yeast Strains, Growth Conditions, and Transformation.The yeast strain C12021, described previously (8), is a trplsucrose-nonfermenting strain suitable for maintaining andexpressing exogenously introduced SUC2 genes. Dere-pressed cells cultured in defined medium containing 0.1%

Abbreviations: S and C invertase, secreted and cytoplasmic forms ofinvertase; ER, endoplasmic reticulum.

5033

The publication costs of this article were defrayed in part by page chargepayment. This article must therefore be hereby marked "advertisement"in accordance with 18 U.S.C. §1734 solely to indicate this fact.

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B AvoHin

EcoRIpAB202

Hind m,Ba 131

PstI and BggXLinkers Added

Deletions Screened

DNA Sequencing ofSmall Deletions

In-Frame" Deletions

Insert TRPI-ARSI Fragmentat EcoRI Site

tructionent I

Yeast Transformation

" In Frame " DeletionsTransformed into Yeast

FIG. 1. Construction of SUC2 signal sequence alterations.(Scheme A) pAB243 contains a 12-base-pair insertion (including aXho I linker) at position + 15 in the former HindIII site within theSUC2 signal sequence coding segment and a single base change atposition -24 in the nearby upstream HindIII site. (Scheme B)pAB202 is a SUC2 precursor plasmid to pAB243 (with the samealteration at position -24) and retains a normal HindIII site in theSUC2 signal sequence coding region. Each of these previouslydescribed plasmids (8) was used to construct deletions as shown inthe schematic diagram. Additional construction details are availableupon request.

glucose and lacking tryptophan were harvested in mid-logarithmic phase.Enzyme Assays and Enzyme Electrophoresis. Assay of S

invertase in whole cells and total invertase (S plus C) inbroken cell extracts has been described (1, 18). Polyacryl-amide gel electrophoresis of invertase was performed -asdescribed (19) except that 0.2% NaDodSO4 was added tosamples, which were then incubated for 5 min at 370C priorto electrophoresis. Continuous recirculation of electropho-retic buffer at 40C ensured enzyme stability.

lInmunoblotting Analysis of Invertase Polypeptides. Invertasepolypeptides were analyzed by transferring from a NaDodSO4/polyacrylamide gel to a nitrocellulose membrane and probingwith invertase antibody and 125I-labeled staphylococcal pro-tein A as described in the legend to Fig. 4.

RESULTS AND DISCUSSION

Construction of Yeast Invertase Signal Sequence Deletions.Previous work suggested that the signal peptide of yeastinvertase is required for secretion of the enzyme (3, 4). Thiswas recently confirmed in vivo by using a SUC2 gene fromwhich a short DNA segment including the signal peptide'sinitiator methionine had been deleted (D.P., P. Brown, P.R.and H.O.H., unpublished data). Although the mRNA for Sinvertase continued to be synthesized with deletion mutantpAB8, it appeared to be translated into a correspondingamount oGnonglyco~lated C enzyme (unpublished data) byutilizing the intact methionine codon at position +61 (5)immediately d&wndtream from the signal sequence. Evidencewas obtained in the same study that the signal sequencesegment of the invertase gene participated in regulating themRNA and thereby enzyme levels for C invertase (mutantspAB9 and pAB223). Given these interesting observations,additional alterations of the signal sequence have beenengineered to study synthetic regulation and secretion of theenzyme.Access to the signal coding region for generating deletion

mutations in plasmid pAB202 was via a natural HindIIIrestriction site (5) located at position + 11 (see Fig. 1, SchemeB, and Fig. 2). Plasmid pAB243, which directed the normalsynthesis, glycosylation, and secretion of invertase (8),contained a Xho I linker inserted at the same HindIII site andwas also utilized for constructing deletion mutations (seeScheme A in Fig. 1). Several of these deletion mutations thatwere sequenced and found to be in-frame (see Fig. 2 forDNAand amino acid sequences) were reintroduced into the su-crose nonfermenting yeast strain CI2021, utilizing autono-mously replicating plasmids (see Materials and Methods).

Synthesis of External and Internal Invertase from SignalSequence Deletions. In yeast cells carrying invertase signalsequence mutations, intracellular invertase activity mayrepresent enzyme that failed to be secreted as well as"genuine" C invertase translated from distinct mRNA tran-scripts. Having generated the altered SUC2 genes shown inFig. 2, we analyzed the resulting intracellular and extracel-lular invertase activities and invertase polypeptides. Theexperiments were designed to distinguish altered enzymesecretion, glycosylation, and signal peptide cleavage func-tions resulting from the mutations.The first experiment described in Table 1 measured extra-

cellular invertase activities found in intact cells and totalactivity found in broken cell extracts. As controls, theparental plasmid, pAB6, and the plasmid pAB8, in which thesignal sequence initiator methionine had been deleted (there-by deleting the entire signal peptide), were included. ThepAB8 transformants synthesized exclusively C invertasepolypeptide (D.P., P. Brown, P.R. and H.O.H., unpublisheddata), in an amount equal to the total invertase ofpAB6. Twoof the deletions, D27 and D36, which retained signal peptidehydrophobic cores of five or fewer amino acids, producednearly twice as much total invertase as the wild-type se-quence yet conferred a Suc- fermentation phenotype to thehost cells. These cells displayed a minor proportion of theirtotal invertase activity as external activity (see Table 1,footnote t). The other two deletions, D49 and D71, whichpreserved longer hydrophobic amino acid cores, conferredSuc+ phenotypes to the cells. D49 produced wild-type levelsof S invertase, whereas D71 produced approximately 4-foldmore S activity. This increased level was similar to that ofpAB273, a homologous plasmid containing a normal signalsequence coding region but an upstream duplication thatincreased invertase mRNA levels and thereby enzyme levels.All of the deletion mutations tested showed levels of intra-cellular invertase significantly elevated over the wild type.

A

EcoRI% pABS

EcoRI

XhoIBal31

XhoI LinkerAdded

Deletions Screened

XhoI, KpnI

KpnXho\,--j

Deletions to 3'Side of Xhol

3 VariableSegments

BamHI,Phosphatase

Xhol,KpnI

5 ReconsSegrm

DNA Sequencing

5034 Biochemistry: Perlman et al.

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Proc. Natl. Acad. Sci. USA 83 (1986) 5035

pAB6 native sequence

met leu leu gin ala phe leu phe leu leu ala gly phe ala ala lys+ ile ser ala + ser metATG CTT TTG CAM GCT TTC CTT TTC1 10 20

CTT TTG GCT GGT TTT GCA GCC MA30 40

ATA50

TCT GCA TCA60

D27-XhoI linkermet leu leu gin

ATG CTT TTG CAM

D36-XhoI linkermet leu leu gin

ATG CTT TTG CAM

ala pro glyGCT CCT CGA GGT -

ala pro glyGCT CCT CGA GGA -

- - gly phe ala ala lys+- - GGT TTT GCA GCC AAA

- - - - - ala lys+- GCC AAM

D49-PstI linkermet leu leu gin leu gin

ATG CTT TTG CAG CTG CAG -

- leu ala gly- CTG GCT GGT

phe al a al a lys+TTT GCA GCC AAA

D71-Bgl II linker

met leu leu his+ ser val

ATG CTT TTG CAC AGA TCT GTC -

leu leu ala gly phe ala ala lys+CTT TTG GCT GGT TTT GCA GCC AAA

FIG. 2. DNA and amino acid sequence oflinker-containing SUC2 signal sequence deletions. The native DNA sequence ofSUC2 determinedby Taussig and Carlson (5), which matched the partial amino acid sequence determined for preinvertase synthesized in vitro (3), is shown atthe top. The signal peptide cleavage site determined for the in vitro products is also shown ( I ). The deletion DNA sequences were obtainedas described in Materials andMethods. Deletions D27 and D36 were obtained by Scheme A (Fig. 1), while D49 and D71 were obtained by SchemeB. The nucleotides contributed by linker insertion and the corresponding amino acids are underlined.

The source of these increased activities will be discussedlater.

Synthesis of Glycosylated and Nonglycosylated Invertase.The major proportion ofinvertase synthesized in derepressedyeast cells is glycosylated and secreted in a process thatfollows insertion of the polypeptide through the endoplasmicreticulum. This secretory pathway in yeast has been definedby a series of host cell mutations (20). If altered invertasesignal peptides were to block secretion before or during ERinsertion, then peptide glycosylation would not be expected.On the other hand, glycosylation might occur if secretionwere blocked at a later stage. We therefore subjected equalamounts of total cell extract (assayed in Table 1) to poly-acrylamide gel electrophoresis, thereby separating the larger,glycosylated, from the smaller, nonglycosylated, invertase(see Fig. 3). The gel, stained for the presence of glucose,shows the separation of activities, the typical downward"smear" ofglycosylated enzyme (which barely enters a 5.5%gel), and a variable upward "tailing" of nonglycosylatedinvertase discussed below. Notably, deletions D27 and D36,which failed to produce extracellular enzyme (Table 1), alsolacked glycosylated invertase (lanes g-l). Therefore wesuspect that the D27 and D36 genetic defects blockedsecretion at a very early stage preceding glycosylation,perhaps blocking polypeptide transport to or through the ERmembrane.A comparison of the relative band intensities of the

glycosylated and nonglycosylated invertases in Fig. 3 withthe relative amounts of S and C enzyme activity in Table 1shows a strong correspondence between the two sets of data(quantitative densitometry could not be obtained with thenonlinear gel staining assay). As for the pronounced tailing ofnonglycosylated invertase, especially apparent with D27 andD36, one possible explanation is offered. Active C invertaseis assembled as a homodimer (21). If secretory processing ofthe signal peptide of preinvertase were blocked and thepolypeptide joined the pool of C invertase, then assembled C

invertase dimers would be heterogeneous in molecularweight. Dimers including uncleaved hydrophobic signal pep-tides would show reduced electrophoretic mobility and,together with normal C invertase dimers, would creategreater electrophoretic heterogeneity.

Immunoblotting Analysis of Invertase Polypeptides. Thesignal sequence deletions D27 and D36 blocked secretion andglycosylation of invertase. However, the elevated intracel-lular nonglycosylated invertase levels that accompaniedthese deletions provided only circumstantial evidence thatpresecretory invertase was still synthesized and contributedactivity to the C enzyme pool. Without evidence thatpreinvertase was translated and maintained in the cell it couldbe argued that the deletions caused an increase in transcrip-tion and subsequent translation ofC invertase. Therefore, animmunoblotting analysis of invertase polypeptides synthe-sized in derepressed cells was conducted to determine thesource of increased C invertase. Yeast cell extracts obtainedby breaking cells and treating with hot detergent wereelectrophoretically fractionated as described in the legend ofFig. 4. A nitrocellulose electroblot of the fractionated pro-teins was probed with invertase antibody and 125I-labeledprotein A to detect invertase polypeptides. The results wereas follows: pAB6, which carries the native signal sequence,and D49 produced the lowest levels of p60. This wasconsistent with the data in Table 1. pAB273, an invertasemRNA overproducing mutant, and D71 both overproduced Sand C invertase, and they showed the highest levels of p60polypeptide synthesis. In contrast to previous reports sug-gesting that C invertase synthesis is constitutive (2, 4), thedata on pAB71 and pAB273 (Table 1, Figs. 3 and 4) provideconvincing evidence that C invertase levels can be modulatedas previously suggested (6). The above constructions con-

tinued to produce glycosylated invertase (Fig. 3). The lasttwo plasmids, D27 and D36, which carried the largest signalsequence coding deletions and which failed to either produceglycosylated invertase or secrete substantial enzyme activi-

ATG

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5036 Biochemistry: Perlman et al.

Table 1. Extracellular and intracellular invertase enzymeactivities produced from altered SUC2 genes shownin Fig. 2

Specific enzymeactivity,

units/1010 cells

Fermentation Whole CellPlasmid and description phenotype* cellst extractt

pAB6,§ wild-type parental Suck 142 82124 82135 75

pAB8, signal peptide not Suc- (7) 220synthesized (7) 178

(11) 241D27 deletion (Xho I) Suc- (28) 408

(28) 469(36) 394

D36 deletion (Xho I) Suc- (32) 277(57) 440(32) 366

D49 deletion (Pst I) Suck 131 160131 142124 121

D71 deletion (Bgl II) Suck 557 650540 525582 632

pAB273, AGCT duplication Suck 458 316at position -27 415 302

543 380For each plasmid, three independent yeast transformants were

cultured and their cell extracts were assayed for invertase (1 activityunit produces 1 /Amol of glucose per min at 30'C, pH 5.5). The sameextracts were electrophoresed (see Fig. 3).*Ability or inability of transformants to ferment sucrose anaerobi-cally (8) is denoted by Suc' or Suc-, respectively.

tExtracellular activity was determined with intact derepressed yeastcells. Numbers in parentheses represent cellular "leakiness" in theassay [typically, 5-1o ofthe intracellular enzyme activity could bedetected with intact cells (D.P., unpublished observations)].tIntracellular activity was determined by subtracting extracellularactivity (column 3) from total activity measured with broken cellextracts. Extracts were prepared from the whole cell suspensions bymixing with glass beads (1). Total extract activity included that dueto cell debris.§S and C invertase activities expressed from linker insertion(nondeletion) "control" plasmids pAB243, -253, and -263 have beenpreviously shown to be comparable to those in pAB6 (8).

ty, were both found to synthesize p62-like invertase species.We have previously demonstrated that pre-S invertase car-rying the signal peptide (synthesized in cell-free translationsystems lacking ER membrane-processing components) mi-grates with an apparent molecular mass of approximately 62kilodaltons in NaDodSO4/polyacrylamide gels (1). The p62-like species in Fig. 4 probably represent invertases withmutant uncleaved signal peptides. The somewhat exaggerat-ed physical separation of the p60- and p62-like speciesobserved in this experiment has been previously noted forplasmid pAB243 (8). pAB243 contains the same Xho I linkerencoding the peptide Pro-Arg-Gly found in D27 and D36. Thepresence of this sequence, predicted to introduce a ,3-turnsecondary structure (8, 23), would reduce the electrophoreticmobility of pre-S invertase consistent with the band separa-tion seen in Fig. 4. Another source of the p62-like species wasalso considered and tested. If the D27 and D36 signal peptidesallowed invertase to begin the process of ER membraneinsertion and glycosylation then the p62 species might arisefrom limited glycosylation of the polypeptide. Endoglycosi-dase H treatment of D27 and D36 proteins was performed as

a bc d e f gh i i k I mno q r

_ sylated... -t-

l.

_ Nongtyco-sylated

FIG. 3. Gel of enzyme activities of extracts from derepressedcells carrying SUC2 variant signal sequences. Transformant cells ofyeast strain 2021 were processed as described in Materials andMethods. Glycosylated and nonglycosylated invertase activities inextracts from approximately 2 x 106 cells were separated in each laneof a 5.5% polyacrylamide gel as shown. Three independent yeasttransformant extracts were analyzed for each genetic construction toensure consistency of plasmid expression. Lanes a-c, pAB273 (anmRNA regulatory mutant to be described elsewhere having anAGCT duplication at position -27 while retaining a native signalsequence); lanes d-f, pAB6 (native SUC2 gene); lanes g-i, D27deletion; lanes j-l, D36 deletion; lanes m-o, D49 deletion; lanes p-r,D71 deletion.

previously described (1) to remove N-linked carbohydratechains. The procedure, however, failed to alter the mobilityofthe p62-like polypeptides, indicating that glycosylation hadnot occurred (data not shown). We propose that p62 synthe-sis is brought about by the failure of the defective signalpeptide to initiate association with the ER membrane.Mechanisms of Regulating S and C Invertase Levels. We

have previously described regulation of S and C invertasemRNA and enzyme levels resulting from glucose derepres-sion, heat shock, and position of the cell in the division cycle(6). cis-acting DNA sequences responsible for invertaseregulation have been studied. For example, DNA sequencesresponsible for glucose regulation of the S invertase mRNAlevel lie 400-500 nucleotides upstream ofthe invertase codingsequence (24). Other regions of the SUC2 gene also affectmRNA levels. For example, increased levels of S and Cinvertase resulted from duplication of an AGCT sequencejust upstream from the signal sequence coding region (D.P.,P. Brown, P.R., and H.O.H., unpublished data). A homol-ogous duplication in the signal sequence coding regionincreased C invertase mRNA and enzyme levels as well.Other constructions such as pAB8, deleting the signal peptideinitiator methionine, helped demonstrate that mRNA thatwas translationally inactive for synthesis of S invertase couldstill be highly effective in directing synthesis of C invertase(via downstream translation initiation). The present experi-ments have suggested that pre-S invertase that fails to besecreted through mutations in the signal peptide region mayalso contribute to the C invertase pool. The intracellularinvertase activity produced from our mutations was variablebut always greater than from the native SUC2 sequence(Table 1). Part of the increase with D27 and D36 can beascribed to the additive contribution of defective secretoryinvertase and the remainder, presumably, to overproductionof C invertase. Regarding D71, we do not presently knowwhether increased p60 synthesis resulted from increasedtranscription or translation. With respect to S invertasesynthesis from D71, it is conceivable that the signal peptidemutation, by introducing two positively charged amino acidsnear the amino terminus, improved cotranslational secretionabove the wild-type level. This suggestion is supported by thefinding that a decrease in the number of positive chargespreceding the hydrophobic core of the X receptor signal

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Proc. Natl. Acad. Sci. USA 83 (1986) 5037

A a b c d e f g h k k I'

~~e..

kDa

68 -

62- ._ v p62

B __p6231 - <~~~~~~~~p60FIG. 4. Immunoblotting analysis of invertase polypeptides in

extracts from derepressed cells carrying variant SUC2 signal se-quences. C invertase (p60) and putative uncleaved pre-S invertasethat failed to be glycosylated and secreted (p62) are shown in thisautoradiogram. NaDodSO4 cell extract supernatants from approx-imately 107 broken cells were electrophoresed in an 8.0%o polyacryl-amide gel. The positions of bovine serum albumin, catalase, andglutamate dehydrogenase standards are indicated on the left. Pro-teins were electrophoretically transferred to nitrocellulose andprobed with polyclonal invertase antiserum (1) plus "2I-labeledprotein A as previously described (22). Additional details areavailable upon request. Lanes a and b, independent yeast transform-ants carrying pAB6; lanes c and d, pAB273 (see Fig. 3 legend); lanese and f, D27; lane g, irrelevant; lanes h and i, D36; lane j, D49; lanesk and 1, D71. Lanes k' and 1' are identical to k and I except that V3as much extract was electrophoresed (see overloading and p60 bandbroadening in lanes k and 1). B is a longer autoradiographic exposureto confirm the single p60 band in lanes k' and 1'.

peptide decreased the cotranslational yield of this secretedprotein in E. coli (25).Our data suggest two functions for the signal sequence

region of SUC2 DNA in yeast. First, this region enablesinvertase to be secreted and, second, it participates inregulating the level of cytoplasmic invertase,

Signal Peptide Structure and Function. As discussed earli-er, a survey of signal peptide structures indicated the pres-ence of 8 or more hydrophobic amino acids within the coreregion. In the construction of previous invertase signalpeptide insertion mutations at the HindIII site at position + 11(8), 11 contiguous hydrophobic amino acids were alwayspresent and secretion function was maintained. From thesecretion phenotypes of the present deletion mutations, wehave learned that the 8 contiguous nonpolar amino acidresidues (Val through Ala, position 19 and following) indeletion D71 suffice for secretion. Moreover, D49, which hasonly 6 contiguous nonpolar residues (between Gln and Lys+),is also secretion-positive. However, we believe that themultiple Leu-Gln residues in D49 located at the aminoterminus of the nonpolar segment adds to the core's contin-uous a-helical or 3strand structure [based on the Chou andFasman structural analysis data (23)]. The deletions D27 andD36 contain no more than 5 hydrophobic residues and, as

expected, were secretion-negative. Clearly a hydrophobiccore of greater than S (probably 8) amino acid residues isrequired for secretion function. The possibility that the XhoI linker in D27 and D36, by introducing the residues Pro-Arg-Gly at the beginning of the hydrophobic core segment,might contribute to the disruption of secretion has also beenconsidered. We believe this possibility is unlikely, however,since pAB243, which contains the same Xho I linker codinginformation preceding a longer hydrophobic core sequence,directs normal synthesis and secretion of invertase (8).Analysis of mutations in the signal peptides of the maltose-binding protein (26) and the X phage receptor protein (27) ofE. coli support the above concepts on structural continuity.In particular, the disruption of hydrophobic core continuityby substitution of charged amino acid residues (includingGlu-, Asp-, Lys', and Arg') prevented the export of boththese proteins. Moreover, after the hydrophobic core of theX receptor signal peptide had been shortened by a 12-base-pair deletion (thus blocking secretion), certain amino acidsubstitution mutations nearby could restore secretion (7).These substitutions (Cys for Gly and Leu for Pro) extendedthe length of the amino acid segments capable of formingcontinuous a-helical or 3-strand structures.

We acknowledge the assistance of W. Theurkauf and the Wensinklaboratory for assistance in establishing procedures for sequencingDNA. This work was supported by a grant from the NationalInstitutes of Health (GM30707) and a grant from the AlfredJurzykowski Foundation.

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