The N-terminus of Sec61p plays key roles in ER protein import and ERAD

Sec61p is the channel-forming subunit of the heterotrimeric Sec61 complex that mediates co-translational protein import into the endoplasmic reticulum (ER). In yeast, proteins can also be post-translationally translocated by the hetero-heptameric Sec complex, composed of the Sec61 and the Sec63 complexes. The Sec61 channel is also a candidate for the dislocation channel for misfolded proteins from the ER to the cytosol during ER-associated degradation (ERAD). The structure of the Sec61 complex is highly conserved, but the roles of its N-terminal acetylation and its amphipathic N-terminal helix are unknown so far. To gain insight into the function of the Sec61p N-terminus, we mutated its N-acetylation site, deleted its amphipathic helix, or both the helix and the N-acetylation site. Mutation of the N-acetylation site on its own had no effect on protein import into the ER in intact cells, but resulted in an ERAD defect. Yeast expressing sec61 without the N-terminal amphipathic helix displayed severe growth defects and had profound defects in post-translational protein import into the ER. Nevertheless the formation of the hetero-heptameric Sec complex was not affected. Instead, the lack of the N-terminal amphipathic helix compromised the integrity of the heterotrimeric Sec61 complex. We conclude that the N-terminal helix of Sec61p is required for post-translational protein import into the ER and Sec61 complex stability, whereas N-terminal acetylation of Sec61p plays a role in ERAD.

Introduction 4 the ER [12]. The cytosolic C-terminus of Sec61p has also been shown to contact the ribosome 66 and is functionally important [13,14]. The cytosolic face of the Sec61 channel also interacts 67 with proteasomes in an ATP-dependent manner [15]. Proteasomes bind the Sec61 channel via 68 the AAA-ATPases of the 19S regulatory particle and in vitro compete with ribosomes for ER 69 membrane binding [16]. The AAA-ATPase Cdc48p, involved in the delivery of both 70 misfolded ERAD substrates and partially translocated proteins to the proteasome, can also 71 bind to the Sec61 channel [2,17]. The specific cytosolic domains of the Sec61 channel 72 responsible for the interaction with AAA-ATPases, however, still remain to be determined 73 [16,18]. The Sec61 complex also interacts with other transmembrane protein complexes via 74 its small subunits: the mammalian orthologue of Sbh1p, Sec61β, mediates interaction with the 75 signal peptidase complex, and its yeast homologue, Sbh2p, binds to the SRP receptor [19,20]. 76 Sbh1p and Sss1p also make contact with the oligosaccharyl transferase complex [21,22].

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Despite the fact that Sec61p structure and function have been extensively characterized, 79 the role of its N-terminus is still unknown. The N-terminal region of Sec61p is likely to be 80 functionally important, given that a 6-histidine tag at the Sec61p N-terminus in combination 81 with point mutations elsewhere in the protein interferes with import, but the phenotype is less 82 severe in the absence of the tag [23]. The N-terminus of Sec61p is oriented towards the 83 cytosol and residues 3-21 have the potential to form an amphipathic α-helix [24]. Together 84 with the Sbh1p N-terminus, the Sec61p N-terminus is exposed at one side of the 85 transmembrane helix bundle forming the transmembrane channel and thus poised to make 86 contact with other proteins [14]. In addition, the starting methionine of Sec61p is cleaved and 87 the serine at position 2 acetylated by the NatA complex [25]. N-terminal acetylation occurs 88 co-translationally, and in yeast about 50% of proteins are N-terminally acetylated, but the 89 significance of this modification is only beginning to be understood [26]. Roles for N-90 5 terminal acetylation include protein stability, interaction ability, and subcellular targeting 91 [27].  The sec61-32 point mutant [28] [31]. Cells were grown in synthetic complete 132 medium without tryptophan to an OD 600 of approximately 0.5 and aliquots of 1 ml were 133 harvested by centrifugation and resuspended in 1 ml of Z buffer (60 mM Na 2 HPO 4 , 40 mM    Lysates were heated for 10 min at 65ºC and analyzed by Western blotting as described above.  In vitro post-translational translocation assay 185 Microsomes were prepared as described in Pilon et al. (1997) [28]. ppαF was translated and    presence and absence of 0.25 μg/ml tunicamycin (Fig. 1C). The strains exhibited severe 256 growth defects under all conditions tested and no growth at all at 24°C in the presence of 257 12 tunicamycin (Fig. 1C). These results indicate that the N-terminal domain of Sec61p is highly 258 important for function.

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To directly investigate UPR induction of the sec61∆H1, sec61∆N21 and sec61S2Y 261 strains, we transformed each of the mutants and the corresponding wildtype with plasmids 262 carrying the β-galactosidase gene under the control of a promoter without or with a UPR 263 element [31]. Transformants were grown at 30°C, lysed, and β-galactosidase activity 264 monitored using a chromogenic substrate [31]. The UPR was strongly induced in the 265 sec61∆H1 strain, but although the β-galactosidase activity was higher in sec61∆N21 than in 266 wildtype cells, this was independent of the UPRE (Fig. 1D). Despite its temperature-and 267 tunicamycin-sensitivity, in the sec61S2Y mutant the UPR was not induced (Fig. 1D).

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Collectively, our data suggest that repositioning of the N-terminal acetylation site in 269 sec61∆H1 is more detrimental to ER protein homeostasis than the absence of N-acetylation in   Fig. 2A).

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In the helix deletion mutants sec61∆N21 and especially sec61∆H1 CPY* accumulated in the 279 ER during the chase, suggesting that post-translational import of CPY* into the ER was still 280 taking place after protein biosynthesis had been inhibited with cycloheximide (Fig. 2B). 281

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Whether or not ERAD was also affected in these mutants remained unclear, even when we 282 extended the chase to 90 min (data not shown). We conclude that N-acetylation of Sec61p at 283 S2 is required for ERAD of misfolded soluble proteins. As the results shown in Fig. 2B suggest an ER import defect or an ERAD defect for 288 sec61∆H1 and sec61∆N21, we decided to investigate import directly. We first monitored co-  Sec61p had no effect on post-translational import of ppαF in vivo (Fig. 3B, left panel). In 302 addition, cytosolic accumulation of ppαF in sec61S2Y was not increased at 37°C, despite the 303 temperature sensitivity of the sec61S2Y mutant (see Fig. 1B). In the cold-sensitive sec61-32 304 control strain, however, ppαF levels were increased at 20°C (Fig. 3B, left panel). As shown in  Post-translational protein import into sec61S2Y, sec61∆H1 and sec61∆N21 microsomes 318 is impaired in vitro 319 As subtle translocation defects can be masked by the abundance of Sec61 channels in intact 320 cells, to further explore a potential impact of the sec61S2Y mutation on protein import into the 321 ER, we investigated the ability of sec61S2Y microsomes to import ppαF in vitro [18,37]. The  (Fig. 4A), and this post-translational import defect was more substantial 327 compared to the one found in intact cells (compare Fig. 4A vs. Fig. 3C, left panel). We also 328 attempted to investigate ppαF import into sec61∆H1 and sec61∆N21 microsomes (Fig. 4B).  To test whether the severe defects in post-translational import found for sec61∆H1 and 338 sec61∆N21 both in vivo and in vitro (Fig. 3 & Fig. 4) were due to an impaired formation of 339 heptameric Sec complexes, we solubilized wildtype, sec61∆H1, and sec61∆N21 microsomes 340 in digitonin, ultracentrifuged lysates, and precipitated Sec complexes from the supernatant 341 using Concanavalin A beads, which bind to the N-glycans of Sec71p [23]. The solubility of 342 Sec61ΔH1p and Sec61ΔN21p was dramatically reduced compared to wildtype, suggesting 343 that Sec61p without its N-terminal helix is prone to aggregation (Fig. 5A, Sol. fractions).

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Although we could only solubilize small amounts of Sec61ΔH1p and Sec61ΔN21p, we were 345 able to detect amounts of both variants in the Con A fractions although the ratios of 346 Sec61ΔH1p and Sec61ΔN21p to Sec63p in the ConA fractions were lower compared to 347 wildtype (Fig. 5A, ConA fractions). In contrast, we observed a dramatic loss of both mutant 348 sec61 protein variants from the ribosome-associated membrane protein (RAMP) fractions 349 (Fig. 5A). We found Sec63p in comparable amounts in the Con-A and RAMP fractions of 350 wildtype, sec61∆H1, and sec61∆N21 membranes, but there was more Sec63p in the "Free" 351 fraction in the mutants compared to wildtype (Fig. 5A, bottom panels). Our data suggest that 352 16 despite the strong post-translational import defects shown by both the N-terminal helix 353 deletion mutants, their heptameric Sec complexes are largely intact.

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By contrast, either the stability of the Sec61 complex or its interaction with ribosomes 356 seemed to be compromised in the absence of the N-terminal helix, as indicated by the reduced 357 amount of mutant Sec61p in the RAMP fractions (Fig. 5A, top panels). Next, we therefore 358 investigated the stabilities of the sec61∆H1 and sec61∆N21 trimeric complexes directly. We 359 solubilized wildtype, sec61∆H1, and sec61∆N21 microsomes in 1% Triton X-100 and 360 centrifuged solubilized Sec61 complexes through a shallow 0-15% sucrose gradient [38].  The Sec61 complex mediates protein import into the ER, and is also a candidate channel for 378 the dislocation of ERAD substrates [3,5]. To investigate the function of the Sec61p N-379 terminus in these processes in yeast, we characterized a set of sec61 N-terminal mutants. We 380 have shown here that N-acetylation of Sec61p at S2 is important for ERAD ( Fig. 2A), and 381 may contribute to post-translational import into the ER (Fig. 4A), whereas its N-terminal 382 amphipathic helix is essential for post-translational import into the ER and is required for 383 stability of the Sec61 complex (Fig. 3C, Fig. 5B).

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Role of Sec61p N-acetylation 386 We investigated Sec61p function in the sec61S2Y mutant, in which Sec61p is not acetylated at 387 its canonical NatA consensus serine at position 2 after methionine cleavage, but rather at the 388 uncleaved initiator methionine, and in two new sec61 mutants: one carrying a deletion of the 389 N-terminal helix but preserving the N-terminal acetylation site in its original sequence 390 context, sec61ΔH1, and one lacking both the N-terminal acetylation site and the N-terminal 391 helix, sec61ΔN21 (Fig. 1A). The mutant strains stably expressed Sec61p at wildtype level 392 (sec61ΔH1) or approximately 40% of wildtype (sec61S2Y and sec61ΔN21, data not shown).

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In a GAL shut-off experiment ER translocation defects only occurred when Sec61p levels fell 394 below 20% of wildtype, and we observed no effect of sec61S2Y on cotranslational and only a does not seem to be the case for our mutant protein (data not shown) [39]. Since mutation of  [40]. 415 Thus, the S2Y mutation will lead to fraying of the N-terminal helix, which is essential for 416 post-translational import (below).
417 418 Fig. 2A shows that CPY* retrotranslocation to the cytosol is considerably delayed in 419 the sec61S2Y mutant. Since the mutant has no protein import defect in vivo (Fig. 3) The sec61∆H1 mutant was the only N-terminal sec61 mutant that displayed a 428 significant UPR induction, although it preserves the N-acetylation site (Fig. 1D). Since the 429 mutant protein is stable, this is unlikely to be a result of Sec61∆H1p eliciting the UPR itself 1C). The growth defect was caused by a strong post-translational import defect (Fig. 3B, 3C, 438 right panels), which -due to the overlap between import and export during the chase -made it 439 difficult to evaluate possible ERAD defects in the helix deletion mutants (Fig. 2B). In 440 addition, we found that all sec61 N-terminal mutants including sec61S2Y were synthetically 441 lethal with an inefficiently translocating post-translational ER import substrate that clogs the 442 channel, suggesting that the Sec61p N-terminus enhances efficiency of post-translational 443 protein import into the ER (data not shown) [42]. the Sec63 complex required for post-translational import is compromised in the absence of 448 the N-terminal helix. We examined this by Con A precipitation of solubilized Sec complexes 449 (Fig. 5A), but to our surprise found no reduction in Sec complex formation in the mutants 450 (Fig. 5A). Instead, we found a dramatic loss of Sec61p from the RAMP fractions of 451 sec61∆H1 and sec61∆N21 membranes, suggesting reduced affinity of mutant Sec61 452 complexes for ribosomes or reduced Sec61 complex stability (Fig. 5A). be compromised if the helix is missing [10]. Given that we observed no DPAPB import 479 defects in sec61∆H1 and sec61∆N21 cells (Fig. 3A, right panel), the Sec61 complex lacking 480 the N-terminal helix seems to be sufficiently stabilized by the ribosome to function during co-481 translational protein import into the ER. Our data suggest that, in the absence of the N-482 terminal helix of Sec61p, the Sec61 complex is unstable, and that its interactions with the 483 Sec63 complex are insufficient to stabilize the channel for post-translational import.