Membrane Topology of the C. Elegans SEL-12 Presenilin

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Neuron, Vol. 17, 1015–1021, November, 1996, Copyright 1996 by Cell Press Membrane Topology of the C. elegans SEL-12 Presenilin

Xiajun Li* and Iva Greenwald*†‡ [this issue of Neuron]; Thinakaran et al., 1996). In the *Integrated Program in Cellular, Molecular, Discussion, we examine the amino acid sequence in and Biophysical Studies light of the deduced topology. † Department of Biochemistry and Molecular Biophysics Results ‡ Howard Hughes Medical Institute Columbia University Sequence analysis suggests that SEL-12 and human College of Physicians and Surgeons presenilins have ten hydrophobic regions (Figure 1). In New York, New York 10032 this study, we provide evidence that a total of eight of these hydrophobic regions function as transmembrane domains in vivo. Below, we use the term “hydrophobic Summary region” to designate a segment of the protein with the potential to span the membrane, as inferred by hydro- Mutant presenilins cause Alzheimer’s disease. Pre- phobicity analysis, and “transmembrane domain” to senilins have multiple hydrophobic regions that could designate a hydrophobic region that our data suggest theoretically span a membrane, and a knowledge of actually spans a membrane. the membrane topology is crucial for deducing the mechanism of presenilin function. By analyzing the activity of ␤-galactosidase hybrid proteins expressed Strategy in C. elegans, we show that the C. elegans SEL-12 We constructed transgenes encoding hybrid SEL- presenilin has eight transmembrane domains and that 12::LacZ proteins, in which LacZ was placed after each there is a cleavage site after the sixth transmembrane of ten hydrophobic regions identified by hydrophobicity domain. We examine the presenilin sequence in view analysis (see Figure 1). We also constructed analogous of the predicted topology and discuss possible mecha- transgenes encoding hybrid SEL-12::TM::LacZ proteins, nisms for presenilin function. which include a short hydrophilic spacer followed by a synthetic transmembrane domain (Fire et al., 1990). Several independent transgenic C. elegans lines ex- Introduction pressing individual LacZ hybrid proteins were established (Mello et al., 1991) and assayed for ␤-galactosi- Mutant presenilin 1 (PS1) and presenilin 2 (PS2) proteins dase activity (Fire, 1992) (Table 1). The transgenes were cause Alzheimer’s disease (Levy-Lahad et al., 1995a, expressed in the pLSX vector (see Experimental Proce- 1995b; Rogaev et al., 1995; Schellenberg, 1995; Sher- dures), which promotes expression in a subset of cells rington et al., 1995). The SEL-12 protein of Caenorhab- that express sel-12. Transgenic nematodes expressing ditis elegans is a true presenilin, based on sequence hybrid proteins in which LacZ is located in the cytosol analysis (Levitan and Greenwald, 1995) and the ability should consistently display staining, but nematodes ex- of PS1 or PS2 to substitute for SEL-12 (see Levitan pressing hybrid proteins in which LacZ is located extra- et al., 1996). The mechanism of presenilin function is cytosolically should not display staining (Silhavy and unknown, but all presenilins have multiple hydrophobic Beckwith, 1985; Froshauer et al., 1988; Fire et al., 1990). regions that could theoretically span a membrane. A Thus, for a pair of hybrid proteins, with the same fusion knowledge of the membrane topology is a crucial step point after a given hydrophobic region but differing only toward deducing the mechanism of presenilin function. in the presence or absence of a synthetic transmem- In this paper, we explore the topology of SEL-12 pre- brane domain, we would expect that one hybrid would senilin in vivo by using a ␤-galactosidase hybrid protein be active but the other would not be active. An example approach that has been widely successful for deducing of this approach for hybrid proteins with a fusion point the topology of membrane proteins (see Froshauer et after the third hydrophobic region is shown in Figure 2. al., 1988; Silhavy and Beckwith, 1985; Traxler et al., 1993; Henn et al., 1995; Lai et al., 1996). This approach relies on the observation that ␤-galactosidase is active within Cytosolic Localization of the Amino and Carboxyl the cytoplasm of cells but not in the extracytosolic com- Termini of SEL-12 Presenilin partment. If ␤-galactosidase is fused at different points Transgenic lines expressing a SEL-12::LacZ hybrid pro- in a membrane protein, its location in the cytosol or in tein in which LacZ is fused near the extreme carboxyl an extracytosolic compartment, and hence the topology terminus, beyond the tenth and last hydrophobic region, of the membrane protein, can be deduced by its activity. display staining, whereas transgenic lines expressing Using the ␤-galactosidase hybrid protein approach, the corresponding SEL-12::TM::LacZ hybrid protein do we have found that SEL-12 presenilin appears to have not display staining. These observations indicate that a total of eight transmembrane domains, with both the the carboxyl terminus of SEL-12 is located in the cytosol. amino and carboxyl termini located in the cytosol. The The SEL-12 presenilin, like PS1 and PS2, does not subcellular localization of hybrid proteins also indicates contain a predicted secretory signal sequence (von that there is an apparent cleavage site after the sixth Heijne, 1986). We have obtained evidence that the amino transmembrane domain. Our results agree with and terminus is cytosolic. First, we examined SEL-12::LacZ complement studies of human PS1 (Doan et al., 1996 and SEL-12::TM::LacZ hybrid proteins and found that Neuron 1016

Table 1. ␤-Galactosidase Activity of SEL-12::LacZ and SEL-12::TM::LacZ Hybrid Proteins Fusion after Number of Lines Fusion Point Hydrophobic Region Reporter Staining/Total

P86 1 LacZ 0/3 1 TM::LacZ 3/4 K129 2 LacZ 3/3 2 TM::LacZ 0/3 S157 3 LacZ 0/5 3 TM::LacZ 5/5 G186 4 LacZ 4/4 4 TM::LacZ 2/4b P211 5 LacZ 0/4 5 TM::LacZ 3/3 G235 6 LacZ 4/4 6 TM::LacZ 0/4 D284 7 LacZ 2/3 7 TM::LacZ 0/3 V389 8 LacZ 0/6 8 TM::LacZ 0/6 P410 9 LacZ 4/4 9 TM::LacZ 0/4 C441 10 LacZ 4/6 10 TM::LacZ 0/6 P410⌬HR8a 9 LacZ 0/8 9 TM::LacZ 4/8

a P410⌬HR8 contains a deletion of amino acids A338–D380. b Two of four lines displayed weak staining, suggesting that the fourth hydrophobic domain may not always span the membrane in the absence of a stabilizing interaction with sequences elsewhere in the protein.

functions as a transmembrane domain, so that the amino terminus of the SEL-12 presenilin is in the cytosol, or the first hydrophobic region does not function as a transmembrane domain, so that the amino terminus is in the extracytosolic compartment. We therefore performed experiments to test whether the first hydrophobic region is able to function as a Figure 1. Hydrophobicity and Amino Acid Sequence Similarity of transmembrane domain. We generated two pairs of con- SEL-12, PS1, and PS2 structs. The first pair added a synthetic signal sequence (A) Hydrophobicity (Kyte and Doolittle, 1982) plot of SEL-12, PS1, (SS) to the SEL-12::LacZ or SEL-12::TM::LacZ hybrid pro- and PS2. A window size of 15 was used. Hydrophobic regions are numbered 1–10. In SEL-12, the hydrophobic regions are bounded tein with a fusion junction after the first hydrophobic region by the following amino acids: 1, L53–Y74; 2, G98–Y123; 3, G133– (HR1). The resulting SS::HR1::LacZ protein displayed Q152; 4, V160–I182; 5, L190–I207; 6, W213–P233; 7, P250–V271; 8, ␤-galactosidase activity, whereas the SS::HR1::TM::LacZ L360–F379; 9, I385–F405; 10, A408–C425. protein did not, indicating that the first hydrophobic region (B) Alignment of the SEL-12, PS1, and PS2 predicted protein se- functions as a transmembrane domain (Table 2). We also quences. The SEL-12 predicted protein sequence shown here is based on Levitan and Greenwald (1995) with a correction of the generated a second pair of hybrid proteins, differing from carboxy-terminal sequence based on information provided by the C. the first pair by the addition of another transmembrane elegans genomic sequencing project (R. Waterston et al., personal domain (HR2, the second hydrophobic region of SEL-12). communication). The PS1 sequence is from Sherrington et al. (1995), Again, the behavior of these proteins indicates that the and the PS2 sequence is from Rogaev et al. (1995). Identical amino first hydrophobic region functions as a transmembrane acids are boxed in black; hydrophobic regions numbered as in Fig- ure 1 are overlined, and the transmembrane domains deduced from domain, since the resulting SS::HR2::HR1::TM::LacZ pro- this study are indicated in Roman numerals as in Figure 4. Closed tein displayed ␤-galactosidase activity, whereas the circles mark fusion points (see Table 1), and closed diamonds mark SS::HR2::HR1::LacZ protein did not (Table 2). the endpoints of the ⌬HR8 deletion (see Table 1). Together, our results indicate that the amino terminus, like the carboxyl terminus, of SEL-12 is cytosolic. An transgenic lines expressing a SEL-12::LacZ hybrid pro- independent study of the topology of human PS1 also tein in which LacZ is located after the first hydrophobic has concluded that the amino and carboxyl termini of region do not display staining, whereas transgenic lines presenilins are cytosolic (Doan et al., 1996). With both expressing the corresponding SEL-12::TM::LacZ hybrid the amino and carboxyl termini located in the cytosol, protein display staining. These observations are consis- SEL-12 and human presenilins must have an even num- tent with two possibilities: the first hydrophobic region ber of transmembrane domains. Presenilin Topology 1017

Figure 2. Strategy for Deducing SEL-12 Pre- senilin Membrane Topology In each panel, the top is a schematic drawing of the location of the LacZ portion of the hybrid protein; a cytosolic location is inferred by the presence of staining, and an extracytosolic location is inferred by the absence of staining. (A) Transgenic hermaphrodite expressing a SEL-12::LacZ hybrid protein with a fusion point at S157 (the third hydrophobic region). (B) Transgenic hermaphrodite expressing a SEL-12::TM::LacZ hybrid protein with a fusion point at S157. This hermaphrodite differs from the hermaphrodite in (A) only in the presence of a short hydrophilic spacer and synthetic transmembrane domain (Fire et al., 1990).

SEL-12 Presenilin Has Eight Transmembrane Domains possibly due to instability of one or both hybrid proteins. The analysis of additional hybrid proteins suggests that Since both the amino and carboxyl termini are located all hydrophobic regions except for the seventh and tenth in the cytosol, indicating that SEL-12 must have an even serve as transmembrane domains, so that the SEL-12 number of transmembrane domains, the hybrid protein presenilin has a total of eight transmembrane domains data were consistent with two possibilities: SEL-12 pre- (Table 1). senilin has a total of eight transmembrane domains, with Transgenic lines expressing SEL-12::LacZ proteins, the eighth and ninth hydrophobic regions (see below) in which the fusion junction occurs after the second, functioning as transmembrane domains, or SEL-12 pre- fourth, and sixth hydrophobic regions displayed stain- senilin has a total of six transmembrane domains, with ing, whereas transgenic lines expressing SEL-12::LacZ only the first six hydrophobic regions functioning as proteins, in which the fusion junction occurs after the transmembrane domains. To distinguish between these first, third, and fifth hydrophobic regions failed to display two possibilities, we compared the activity of hybrid staining. The reciprocal results were observed for SEL- proteins in which LacZ or TM::LacZ is fused after the 12::TM::LacZ hybrid proteins. These results indicate that ninth hydrophobic region in the presence or absence of the first six hydrophobic regions function as transmem- the eighth hydrophobic region (P410 versus P410⌬HR8, brane domains. Table 1). In this case, deletion of the eighth hydrophobic When the fusion junction occurs after the seventh region affected the ␤-galactosidase activity of otherwise hydrophobic region, we observed staining in transgenic equivalent hybrid proteins: the P410::LacZ hybrid is ac- lines expressing SEL-12::LacZ proteins but not SEL- tive whereas the P410⌬HR8::LacZ hybrid is not active, 12::TM::LacZ proteins. Since this pattern is similar to and the P410⌬HR8::TM::LacZ hybrid is active whereas that seen for hybrid proteins in which the fusion junction the P410::TM::LacZ hybrid is not active. These results occurs after the sixth hydrophobic region, we infer that suggest that the eighth hydrophobic region does indeed the seventh hydrophobic region does not function as a function as a transmembrane domain. transmembrane domain, or that it spans the membrane As described above, transgenic lines expressing SEL- twice (see Discussion). 12::LacZ proteins, but not SEL-12::TM::LacZ proteins, The SEL-12::LacZ or SEL-12::TM::LacZ hybrid proteins in which the fusion junction occurs after the tenth hy- were not informative for hybrids in which the fusion drophobic region displayed staining, establishing that junction occurs after the eighth hydrophobic region: we the carboxyl terminus of SEL-12 presenilin is cyto- did not find convincing staining in any lines (Table 1), plasmic. The same results were obtained for transgenic lines in which the fusion junction occurs after the ninth hydrophobic region (i.e., SEL-12::LacZ proteins display Table 2. The First Hydrophobic Region of SEL-12 Functions as ␤-galactosidase activity, but SEL-12::TM::LacZ proteins a Transmembrane Domain do not). From these results, we infer that the ninth hy- Number of Lines drophobic region functions as the final transmembrane Hybrid Staining/Total domain and that the tenth hydrophobic region, like the SS::HR1::LacZ 4/4 seventh hydrophobic region, does not function as a SS::HR1::TM::LacZ 0/4 transmembrane domain (or spans the membrane twice). SS::HR2::HR1::LacZ 0/5 SS::HR2::HR1::TM::LacZ 7/8 Abbreviations: SS, synthetic signal sequence; HR1, first hydropho- SEL-12 Presenilin Is Cleaved after the Sixth bic region; HR2, second hydrophobic region; TM, synthetic trans- Transmembrane Domain membrane domain. For details about these hybrid proteins, see Changes in the subcellular localization of SEL-12::LacZ Experimental Procedures. fusion proteins suggested that the SEL-12 presenilin, Neuron 1018

Figure 3. Deducing the Cleavage Site in SEL- 12 Presenilin Hermaphrodites were stained with DAPI to visualize nuclei as well as for ␤-galactosidase activity (Fire et al., 1990). In each panel, the top is a schematic drawing of the result, with the subcellular localization of ␤-galactosidase staining indicated by grey shading, and the bottom shows a photomicrograph, with the nucleus indicated by an arrow. (A) Transgenic hermaphrodite expressing a SEL-12::LacZ(NLS) hybrid protein with a fusion point at G235 (after the sixth hydrophobic region) shows cytoplasmic staining. (B) Transgenic hermaphrodite expressing a SEL-12::LacZ(NLS) hybrid protein with a fusion point at D284 (after the seventh hydrophobic region) shows nuclear staining.

like PS1 (Thinakaran et al., 1996), is cleaved. In C. ele- Discussion gans, secreted and transmembrane LacZ hybrid proteins are typically localized to the perinuclear region, Topology of SEL-12 Presenilin apparently because secreted or transmembrane forms Using the ␤-galactosidase hybrid protein approach, we of ␤-galactosidase are retained in the endoplasmic retic- have deduced the SEL-12 presenilin topology shown in ulum (Fire et al., 1990). (This property does not affect Figure 4. The inferred topology reveals that in general the the determination of topology based on ␤-galactosidase cytosolic side tends to have more positive charges than activity of hybrid proteins, since the lumen of the endo- the extracytosolic side, consistent with “rules” derived plasmic reticulum is topologically extracytosolic and from analyzing many membrane proteins (Reithmeier, fails to support ␤-galactosidase activity.) In most trans- 1995). Our topological model for SEL-12 presenilin is in genic lines, ␤-galactosidase staining appears largely accord with data for human PS1 (Doan et al., 1996), further perinuclear, consistent with retention in the endoplas- underscoring the conclusion from functional evidence that mic reticulum. However, for the set of transgenic lines SEL-12 is a bona fide presenilin (Levitan et al., 1996). The that express a hybrid SEL-12::LacZ protein in which high degree of sequence conservation apparent when the LacZ is fused after the seventh hydrophobic region, we deduced transmembrane domains of SEL-12 presenilin noticed that staining is not confined to the perinuclear are compared with human PS1 and PS2 (see Figure 1) region and instead was observed throughout the cytoplasm (data not shown). This diffuse staining suggested that in these lines, ␤-galactosidase diffuses away from the membrane, as would be expected if there were a cleavage site prior to the fusion point. To visualize the subcellular localization of LacZ more clearly, we constructed SEL-12::LacZ hybrid proteins in which the LacZ protein also contains a nuclear localization sequence (NLS) (Fire et al., 1990; see Figure 3). As before, if LacZ were to be associated with the membrane, staining would be perinuclear. However, if a cleavage event were to release LacZ from the SEL-12 presenilin membrane tether, then LacZ would enter the Figure 4. Inferred Topology of SEL-12 nucleus and staining would be nuclear. We found that Hydrophobic regions are indicated by Arabic numerals, and trans- the SEL-12::LacZ(NLS) hybrid protein is perinuclear membrane domains deduced from this study are indicated by Ro- when the fusion junction is located after the sixth hyman numerals. As detailed in the Discussion, the eight transmem- drophobic region (at amino acid 235), but is nuclear brane domains may associate with each other to form a ␤ barrel, when the fusion junction is located after the seventh and the seventh and tenth hydrophobic regions may be associated hydrophobic region (at amino acid 284) (Figure 3). These with the membrane (represented by a double-headed arrow), with observations suggest that there is a cleavage site be- other domains of SEL-12, or with other proteins. The deduced cleavage site is likely to be within amino acids G235 to D284; for simplicity tween (or near) amino acids 235 and 284 and that se- in this diagram, the uncleaved form is shown. Cleavage does not quences beyond amino acid 284 are not needed for the appear to be absolutely required for PS1 function in a C. elegans cleavage event. functional assay (Levitan et al., 1996). Presenilin Topology 1019

suggests that the human presenilins have corresponding Interactions among transmembrane domains may transmembrane domains. form a passageway through the membrane. Thus, it is The presence of many potential transmembrane do- conceivable that presenilins function as, or as part of, mains in presenilins led to speculations about potential a channel, transporter, or pore. Interactions among functions for presenilins as G protein–coupled recep- ␣-helical transmembrane domains may form a channel, tors, channels, and docking proteins, participating in as has been seen for bacteriorhodopsin, in which five processes as diverse as protein trafficking, signal recep- of the seven ␣ helices form a proton channel (Henderson tion, and chromatin binding (e.g., Clark et al., 1995; Levi- et al., 1990). Alternatively, interactions among ␤ strands tan and Greenwald, 1995; Levy-Lahad et al., 1995b; Li may form a ␤ barrel, a structure that has been proposed et al., 1995; Rogaev et al., 1995; Sherrington et al., 1995). for a variety of pores, transporters, and channels, and We have found that SEL-12 presenilin appears to have has been confirmed by three-dimensional structural a total of eight transmembrane domains, with both the analysis for porins and ␣-hemolysin (Cowan et al., 1992; amino and carboxyl termini located in the cytosol. Since Song et al., 1996). The large number of consecutive SEL-12 has eight transmembrane domains and appears hydrophobic amino acids in each individual transmem- to be cleaved into proteins with six and two transmembrane domain do not resemble the alternating charged brane domains, presenilins do not at this level resemble or polar residues typical of putative ␤ barrel pores that canonical seven transmembrane domain G protein– pass hydrophilic molecules, and the rarely interspersed coupled receptors. Instead, the presenilins appear to polar or charged amino acids do not occur at intervals be a novel class of multipass membrane protein. characteristic of an amphipathic ␣ helix. Our data suggest that SEL-12 presenilin is cleaved after the sixth transmembrane domain, as has also been observed for human PS1 (Thinakaran et al., 1996). Cleav- Other Presenilin Domains age does not appear to be absolutely required for pre- Most of the intertransmembrane sequences are short senilin function, since a PS1 mutant that lacks the cleav- and very highly conserved among all three presenilins. age site is able to function in place of SEL-12 in C. The main cytosolic domains are the amino and carboxyl elegans (Levitan et al., 1996). The amino- and carboxy- termini, and the large “loop” between the sixth and sev- terminal cleavage products of PS1 are present in a 1:1 enth transmembrane domains. There are no large extra- stoichiometry (Thinakaran et al., 1996). However, it is cytosolic domains. There is a single conserved potential not known if the cleavage products function together or N-linked glycosylation site, located in the extracellular have independent activities; it is also not known whether domain between the seventh and eighth transmembrane they oligomerize or associate with other membrane pro- domains. teins. The amino-terminal domain is not well conserved Localization studies have suggested that presenilins among all three presenilins, except for a very short are found in intracellular membranes, with little or no stretch of about ten amino acids immediately before the detectable presenilin protein in the plasma membrane first transmembrane domain. The amino-terminal region (Kovacs et al., 1996). Together, the localization and topo- of PS2 resembles a PEST sequence, which has been logical information is consistent with a function for pre- implicated in promoting protein turnover (Rogers et al., senilins in the biogenesis or transport of membrane pro- 1986; Rechsteiner, 1990), but the amino termini of SEL- teins, or in facilitating communication between different 12 and PS1 are not clearly recognizable as PEST se- subcellular compartments. quences. The carboxy-terminal domain is well conserved, particularly in the tenth hydrophobic region, in which almost every amino acid is identical among all Presenilin Transmembrane Domains three presenilins. This domain might mediate protein– The high degree of sequence conservation among SEL- protein interactions within or between presenilin mole- 12, PS1, and PS2 suggests that the transmembrane cules, or with other proteins. Alternatively, it may be domains may form a conserved structure and have con- directly associated with the lipid bilayer. served interactions with each other or with other mole- The seventh hydrophobic region deduced from hy- cules. dropathy analysis does not appear to span the mem- Individual SEL-12 transmembrane domains are about brane and is instead part of a large intracellular “loop” 20 amino acids long. They are thus long enough to span between the sixth and seventh transmembrane do- a lipid bilayer in an ␣-helical conformation, as has been mains. If the transmembrane domains of presenilins observed for the transmembrane domains of multipass form a pore, then it is conceivable that the intracellular membrane proteinssuch as bacteriorhodopsin (Hender- loop (and/or tenth hydrophobic region) intrudes into the son and Unwin, 1975; Henderson et al., 1990) and cyto- pore and functions as part of a gating mechanism chrome c oxidase (Iwata et al., 1995). Alternatively, it is (MacKinnon, 1995). (This function may still occur in the possible that some or all of the transmembrane domains cleaved molecule, if the cleavage products remain asso- of SEL-12 presenilin form ␤ strands. A standard 2Њ struc- ciated.) ture prediction algorithm (Chou and Fasman, 1978) pre- The postulated loop appears to have twosubdomains. dicts that the deduced transmembrane domains of SEL- One subdomain begins after the sixth transmembrane 12 presenilin would have a propensity to form ␤ strands domain and includes the seventh hydrophobic region, rather than ␣ helices, although we note that this algo- and is highly conserved among all three presenilins. This rithm may not be valid for transmembrane domains in subdomain may encompass the cleavage site (Thina- a bilayer. karan et al., 1996; this study). As is the case for the tenth Neuron 1020

hydrophobic region, the seventh hydrophobic region Plasmids may interact with the membrane or mediate protein– To construct plasmids encoding the hybrid proteins shown in Table protein interactions. Alternatively, if the seventh hy- 1, we first constructed pLSX, an expression vector containing 2.8 kb of sel-12 5Ј flanking region, a polylinker, and 755 bp of 3Ј noncoding drophobic region remains intact and adopts a ␤ strand region, including a polyadenylation sequence, from the unc-54 gene conformation, it would be theoretically long enough to (Fire et al., 1990). This vector leads to good expression in a subset span the membrane twice, in a hairpin-like structure, of cells that express sel-12 (X. L., D. Levitan, and I. G., unpublished and hence would not behave like a membrane-spanning data). cDNAs containing initiation codons are inserted into the poly- domain in the LacZ hybrid protein assay. At least part linker site, and expression is driven by sel-12 5Ј flanking region. of the seventh hydrophobic region can be deleted from Portions of sel-12 cDNA (Levitan and Greenwald, 1995) were ampli- fied by the polymerase chain reaction (PCR) and fused in-frame to PS1 without abolishing its ability to substitute for SEL- the cDNAs encoding LacZ proteins, with or without an upstream 12 in a C. elegans functional assay (Levitan et al., 1996), synthetic transmembrane domain (Fire et al., 1990). The resultant suggesting that this part of the “loop” is dispensible for sel-12::lacZ and sel-12::tm::lacZ hybrid cDNAs were placed into normal function. However, it is possible that the mutant pLSX. PS1 protein, which can cause Alzheimer’s disease The plasmids encoding the hybrid proteins shown in Table 2 are (Perez-Tur et al., 1995), may have a novel gain-of-func- also involved insertion into pLSX. SEL-12 sequences were generated by PCR, and insert coding regions were assembled in pBlue- tion activity. script (Stratagene) prior to insertion into pLSX. “SS”, the synthetic The second subdomain of the intracellular loop is es- signal sequence, is described by Perry et al. (1993); “HR1” encom- sentially unconserved among all three presenilins. How- passes amino acids P2-P86; “HR2” encompasses amino acids S75- ever, in all three presenilins this region strongly resem- K129. bles a PEST sequence (Rogers et al., 1986; Rechsteiner, The integrity of all PCR products was confirmed by sequencing. 1990). It is possible that the level of the proteolytically processed presenilin fragments is regulated by the Sequence Analysis The Genetics Computer Group Wisconsin software package version PEST system for protein degradation. 8 was used for sequence analysis.

Presenilin Topology and Alzheimer’s Disease Acknowledgments In the context of the possibility that presenilins function We thank Denise Brousseau for help with microinjection andRichard as, or as part of, a channel, transporter, or pore, it is worth Ruiz for help with plasmid preparations. We are grateful to Eric considering potential consequences of the amino acid Gouaux and David Hirsh for valuable insights into presenilin topol- changes that cause Alzheimer’s disease. Mutations that ogy and structure and appreciate the helpful comments on the alter the transmembrane domains or juxtamembrane re- manuscript from Richard Axel, Eric Gouaux, Barth Grant, David gions may affect the properties of the opening, or the Hirsh, Jane Hubbard, and Diane Levitan. We also thank Sangram interaction with entities that pass through the opening. Sisodia for communicating results prior to publication. I. G. is an Associate Investigator of the Howard Hughes Medical Institute. Notably, about a third of the known mutations alter amino The costs of publication of this article were defrayed in part by acids in the first subdomain of the postulated loop and the payment of page charges. This article must therefore be hereby hence may alter the gating mechanism. An important chal- marked “advertisement” in accordance with 18 USC Section 1734 lenge for the future is to ascertain how mutant presenilins solely to indicate this fact. increase the extracellular concentration of the A␤1–42(43) peptide (Scheuner et al., 1996; Thinakaran et al., 1996), Received August 29, 1996; revised October 18, 1996. an amyloid precursor protein cleavage product that is References presumed to be the causeof Alzheimer’s disease in mutant individuals. Additional genetic and functional studies of Brenner, S. (1974). The genetics of Caenorhabditis elegans. Genetics SEL-12, including the identification of other genes that 77, 71–94. function in SEL-12-mediated processes, may reveal the Chou, P.Y., and Fasman, G.D. (1978). Prediction of the secondary normal function of presenilins and the aberrant function structure of proteins from their amino acid sequence. Adv. Enzymol. of mutant presenilins. 47, 45–148. Clark, R.F., Hutton, M., Fuldner, R.A., Froelich, S., Karran, E., Talbot, C., Crook, R., Lendon, C., Prihar, G., He, C., Korenblat, K., Martinez, Experimental Procedures A., Wragg, M., et al. (1995). The structure of the presenilin 1 (S182) gene and identification of six novel mutations in early onset AD Genetic Methods families. Nature Genet. 11, 219–222. Methods for handling and culturing C. elegans have been described Cowan, S.W., Schirmer, T., Rummel, G., Steiert, M., Ghosh, R., Paup- by Brenner (1974). The wild-type parent for all strains used was C. tit, R.A., Jansonius, J.N., and Rosenbusch, J.P. (1992). Crystal struc- elegans var. Bristol strain N2 (Brenner, 1974). Strains were grown tures explain functional properties of two E. coli porins. Nature 358, at 25ЊC, a temperature that maximizes ␤-galactosidase activity (J. 727–733. Way, personal communication). Doan, A., Thinakaran, G., Borchelt, D.R., Slunt, H.H., Ratovitsky, T., Transgenic lines were established by microinjection of plasmid Podlisny, M., Selkoe, D.J., Seeger, M., Gandy, S.E., Price, D.L., and mixtures into the hermaphrodite germline to create extrachromo- Sisodia, S.S. (1996). Protein topology of presenilin 1. Neuron 17, somal arrays (Mello et al., 1991). Plasmids were injected at a concen- this issue. tration of 20 ␮g/ml into recipient N2 hermaphrodites along with 100 ␮g/ml of pRF4, a plasmid containing the cloned dominant rol- Fire, A. (1992). Histochemical techiniques for locating Escherichia 6(su1006) gene (Mello et al., 1991), as a cotransformation marker. F1 coli ␤-galactosidase activity in transgenic organisms. Genet. Anal. Roller progeny were picked, and F2 Roller progeny used to establish Tech. Appl. 9, 151–158. lines. Larvae were fixed using an acetone fixationprotocol described Fire, A., Harrison, S.W., and Dixon, D. (1990). A modular set of by (Fire, 1992) and stained for ␤-galactosidase activity overnight at lacZ fusion vectors for studying gene expression in Caenorhabditis room temperature. elegans. Gene 93, 189–198. Presenilin Topology 1021

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