Human Sec62 and Sec63 Interact with Each Other in a Manner Similar to their Yeast Orthologues
In yeast, the negatively charged carboxyterminus of Sec63p interacts with the overall positively charged aminoterminal (N-terminal) domain of Sec62p (Wittke et al., 2000
; A). Thus, we asked if this mode of interaction is conserved in the two human proteins. Purified GST hybrid proteins that comprised the complete carboxyterminal (C-terminal) domain of human Sec63 (termed Sec63C) or only its 26 C-terminal amino acid residues (Sec63C26) were immobilized on GSH-Sepharose. GST served as a negative control. Then, buffer or purified hexa-histidine fusion of the N-terminal domain of mammalian Sec62 (Sec62N) were applied to the resin. The bound material was eluted and analyzed by SDS-PAGE, followed by either protein staining (A, lanes 1 and 2, 4 and 5, and 7 and 8) or Western blotting plus immunodetection with anti-penta-histidine antibodies (B). Although there was no Sec62N detected in the eluate of the immobilized GST (lane 2), there was a significant amount of Sec62N found in the eluate of both GST-Sec63C (lane 5) as well as GST-Sec63C26 (lane 8). Thus, human Sec63 and Sec62 directly interact with each other, and the C-terminal 26 amino acid residues of Sec63C are sufficient for this interaction.
Figure 2. Human Sec62N interacts with human Sec63C. (A and B) GST or the two GST hybrid proteins, GST-Sec63C or GST-Sec63C26, were immobilized on GSH-Sepharose. Buffer, Sec62N, or Sec62N-ΔN10-ΔC40 (1 μM) was applied to the resin. After washing, (more ...)
We analyzed which part of Sec62N was involved in this interaction: the central and overall positive region or one of the two positively charged oligopeptides at/near the amino- or carboxyterminus (, A and B). This was addressed by employing a truncated Sec62N that lacks the two positively charged oligopeptides (termed Sec62N-ΔN10-ΔC40; C) or the two positively charged oligopeptides (termed 62-11mer1 and 2; C) as potential competitors of Sec62N, respectively. Sec62N-ΔN10-ΔC40 efficiently bound to GST-Sec63C as well as GST-Sec63C26 (, A and B, lanes 6 and 9). Furthermore, even at a >100-fold molar excess (that is effective in the competition of ribosome binding; see below) the two basic oligopeptides did not interfere with binding of Sec62N to GST-Sec63C (C). Thus the central and overall positive region of Sec62 is involved in interaction with Sec63C. This was confirmed by binding studies that used peptide spot membranes according to Frank (1992)
(Supplemental Figures 3 and 4). In this respect, both mammalian proteins behaved like their yeast orthologues.
To further characterize the Sec62/Sec63 interaction, SPR experiments were carried out. Human Sec62N was immobilized in the measuring cell of a NTA sensor chip via its hexa-histidine tag. Human Sec62C served as a negative control and was immobilized in the reference cell. Then increasing concentrations of human Sec63C were passed over the chip and were followed by buffer. Association of the analyte and its dissociation were recorded and analyzed (D). We could fit the kinetics with a 1:1 binding model and determined an apparent affinity (Kd
) of Sec63C for Sec62N of 4.78 nM. This Kd
was consistent with the fact that native Sec62 was coimmunoprecipitated with Sec63 from a microsomal detergent extract (Tyedmers et al., 2000
Human Sec62 Interacts with 80S Ribosomes
The N-terminal domain of mammalian Sec62 (Sec62N) contains two basic oligopeptide motifs that are reminiscent of similar peptides in established ribosomal tunnel exit ligands (such as NAC and ERj1; Ferbitz et al., 2004
; Blau et al., 2005
; B). Deletion of the basic oligopeptides in the N-terminal domains of mammalian and yeast NACβ (Grallath et al., 2006
; Wegrzyn et al., 2006
) as well as the cytosolic domain of ERj1 (Dudek et al., 2005
) led to loss of ribosome-binding ability. Therefore, Sec62N was analyzed with respect to its ribosome-binding ability by a number of different experimental approaches, and the role of the two basic oligopeptide motifs was characterized. The same experimental strategies were previously used for the characterization of the ribosome interaction of ERj1 (Dudek et al., 2002
Sec62N was incubated in the presence or absence of nontranslating 80S ribosomes. An aliquot of ribosomes was incubated in the absence of Sec62N and served as reference (A). The samples were analyzed by gradient centrifugation and subsequent SDS-PAGE and protein staining. In the absence of ribosomes, Sec62N stayed at the top of the gradient (B). After incubation with ribosomes, Sec62N comigrated with ribosomes (C). Thus the observed comigration of Sec62N with ribosomes was not due to aggregation, but rather reflected an interaction between the two molecules.
Figure 3. Ribosome binding of human Sec62N and inhibition by basic oligopeptides. (A–C) Sec62N was adjusted to KCl concentration of 100 mM and incubated without (B) or with ribosomes (C). A third mixture contained ribosomes but was free of Sec62N (A). Subsequently (more ...)
Next, Sec62N and three truncated derivatives (C) were each incubated in the presence or absence of nontranslating ribosomes in order to address the question of which part of Sec62N was involved in this interaction. Yeast Sec62N that lacks similar basic oligopeptide motifs as compared with human Sec62N (A) served as negative control. Subsequently, the ribosomes were reisolated by centrifugation and the relative amount of ribosome associated Sec62N was determined by SDS-PAGE and protein staining (D) or Western blotting plus immunodetection with anti-penta-histidine antibodies (, E and F). All proteins stayed in the supernatant in the absence of ribosomes (F). However, Sec62N and constructs that contained at least a single positively charged oligopeptide were pelleted together with ribosomes, i.e., were active in ribosome binding (although to a varying degree), whereas the construct with the double deletion (termed Sec62N-ΔN10-ΔC40) was almost completely inactive in ribosome binding (, D and E, lanes 7 and 8). Furthermore, yeast Sec62N that lacks similarly charged oligopeptides was unable to bind ribosomes (, D and E, lanes 9 and 10). Although both basic oligopeptides within Sec62N contributed to ribosome binding, the aminoterminal peptide seemed to be more important.
To further substantiate the role of the two highly charged oligopeptides for ribosome binding, synthetic oligopeptides were used as potential competitors of Sec62N in ribosome binding (peptides 62-11mer1 and 2; C). The aminoterminal oligopeptide from an invertebrate Sec62 served as negative control (peptide iv62-11mer). Only the aminoterminal peptide from human Sec62 competed with Sec62N for ribosome binding (G, lanes 3 and 4).
Having observed an interaction of human Sec62N with ribosomes, i.e., a gain of function of the human protein compared with the yeast protein, we asked if yeast Sec62N can be turned into a ribosome-binding protein by the addition of the aminoterminal dodecapeptide from human Sec62 and if human SEC62
is able to rescue the thermosensitive growth phenotype of a translocation-deficient yeast SEC62
mutant. Yeast Sec62N was extended at its aminoterminus by the aminoterminal dodecapeptide from human Sec62 (termed humanized Sec62N) and incubated in the presence or absence of nontranslating ribosomes. Subsequently, the ribosomes were reisolated by centrifugation and the relative amount of ribosome associated humanized Sec62N was determined by SDS-PAGE and protein staining or Western blotting plus immunodetection with anti-penta-histidine antibodies. In contrast to wild-type yeast Sec62N, humanized Sec62N bound to ribosomes (, H–J). Thus the aminoterminal peptide that is present in human Sec62 is sufficient for ribosome binding. Because of a protein translocation defect at the nonpermissive temperature, the yeast mutant strain RDM50-94C hardly grows at 37°C (Deshaies and Schekman, 1990
). However, when the human SEC62
gene was expressed in this strain after the addition of galactose, the cells grew at 37°C (). Thus, the additional positively charged oligopeptides that are present in human Sec62 compared with yeast Sec62p do not interfere with the posttranslational function of yeast Sec62p.
Figure 4. Human Sec62 complements a corresponding yeast mutant strain. Yeast SEC62 ts mutant strain RDM50-94C was grown at 24 or 37°C in the absence of uracil and in the presence of glucose or galactose as indicated. The cells had been transformed with (more ...)
To quantitatively characterize the ribosome interaction of mammalian Sec62, SPR experiments were carried out. Human Sec62N was immobilized in the measuring cell of a NTA sensor chip via its hexa-histidine tag. Based on the experiments that are depicted in , D and E, yeast Sec62N served as a negative control and was immobilized in the reference cell. Then increasing concentrations of mammalian ribosomes were passed over the chip and were followed by buffer. Association of the analyte to and its dissociation from the ligand were recorded and analyzed (A). We determined an apparent affinity (Kd) of ribosomes for Sec62N of 0.13 nM.
Figure 5. Characterization of the Sec62/ribosome interaction. (A) Human Sec62N was immobilized on an activated NTA sensor chip in the measuring cell and yeast Sec62N as a negative control in the reference cell. Increasing concentrations of nontranslating ribosomes (more ...)
Ribosome Association of Sec62 Is Salt- and RNase-sensitive
The interaction of ERj1 with ribosomes was shown to be salt-sensitive and to involve rRNA (Dudek et al., 2002
). Here, we asked if this is also true for Sec62N. Thus, ribosome binding assays and SPR experiments were carried out above the standard 100–150 mM salt concentration and by substituting ribosomes by RNase-treated ribosomes, respectively. Human Sec62N was immobilized in the measuring cell of a NTA sensor chip via its hexa-histidine tag, and yeast Sec62N was immobilized in the reference cell. After association of native ribosomes, the dissociation was carried out at 200 and 500 mM KOAc concentration (B). According to the sensorgram, the interaction of Sec62N with ribosomes was salt-sensitive. When RNase treated ribosomes were used as analyte the sensorgram suggested that the interaction of Sec62N with ribosomes was RNase-sensitive (C). Similar observations were made in the ribosome-binding assays (, D and E vs. F). Thus binding of Sec62N to ribosomes most likely involves electrostatic interactions with rRNA. We note that the observed salt sensitivity of ribosome binding of Sec62N may explain why Sec62 was not characterized as ribosome-associated membrane protein after solubilization of microsomes in detergent (salt concentration: 400 mM KCl; Meyer et al., 2000
; Tyedmers et al., 2000
Sec62 Interacts with Ribosomes near or at the Tunnel Exit
Next, we used three independent experimental approaches to assess whether or not the ribosomal exit site of nascent polypeptides is also the binding site for Sec62N, because this site has previously been observed to provide a docking site for several proteins that are involved in protein biogenesis at the ER (SRP, NAC, Sec61 complex, ERj1; Beckmann et al., 2001
; Halic et al., 2004
; Ferbitz et al., 2004
; Blau et al., 2005
First we assessed if there was a preference of Sec62N for the large ribosomal subunit. Sec62N was incubated in the presence or absence of 60S and 40S ribosomal subunits. The samples were analyzed by gradient centrifugation and subsequent SDS-PAGE as followed by Western blotting plus immunodetection with anti-penta-histidine, anti-L4, or anti-S3 antibodies, respectively. After incubation with 60S subunits, a fraction of the Sec62N comigrated with ribosomes (B). In the absence of ribosomes and in the presence of 40S subunits, Sec62N stayed at the top of the gradient (, A and C). Thus, Sec62 can only interact with large ribosomal subunits.
Figure 6. Sec62N binds near the ribosomal tunnel exit. (A–C) Sec62N was incubated in the absence (A) or presence of 60S (B) or 40S (C) ribosomal subunits. Subsequently the samples were subjected to sucrose gradient centrifugation. The fractions were analyzed (more ...)
Next, we analyzed the possible overlap in binding sites between Sec62N and ERj1 using SPR experiments (Blau et al., 2005
). Sec62N was immobilized in the measuring cell of a NTA sensor chip via its hexa-histidine tag and analyzed with respect to binding of native ribosomes that had been preincubated with ERj1C (D). Native ribosomes that had been preincubated with Sec62N served as positive control, and native ribosomes that had been preincubated with BSA as negative control. The results demonstrate that Sec62N and ERj1 compete for an overlapping binding site on ribosomes, most likely at or near the ribosomal tunnel exit.
To directly address this point, Sec62N or its derivatives were allowed to form complexes with ribosomes that contained radiolabeled nascent polypeptide chains of defined length (i.e., peptidyl-tRNAs comprising 86 amino-terminal amino acid residues of preprolactin, ppl86mer
). Subsequently, the ribosome/nascent chain/Sec62N complexes were reisolated and subjected to chemical cross-linking. ERj1 served as a positive control (Dudek et al., 2005
; E, lane 6). In the presence of Sec62N or a construct that contains a single positively charged oligopeptide, cross-linked products of the nascent polypeptide were detected (E, lanes 2–4). These were absent when buffer, Sec62N-ΔN10-ΔC40 (E, lanes 1 and 5), yeast Sec62N (F, lane 2), or human Sec62C were used (F, lane 3). Thus, like ERj1, Sec62N binds to the ribosome near the tunnel exit. We note that there is a dominant 45-kDa cross-linking product to be seen in the absence of Sec62N that is hardly seen in the presence of Sec62N. We suggest that this cross-linking product involves a 36-kDa ribosomal protein (possibly L4; Woolhead et al., 2004
) and that Sec62N that is bound near the tunnel exit affects the positioning of the nascent polypeptide chain with respect to this ribosomal protein. According to this interpretation, removal of the aminoterminal oligopeptide has a more pronounced effect on binding of Sec62N compared with removal of the carboxyterminal oligopeptide.
Sec62N Does Not Simultaneously Interact with Sec63C and Ribosomes
Having seen an interaction of Sec62N with ribosomes () and with Sec63C (), we asked if Sec62N can recruit Sec63C to ribosomes. In ribosome binding assays, however, we failed to detect a trimeric complex (data not shown). Therefore, the immobilized complex between GST-Sec63C and Sec62N was used (, left panels). In parallel, a complex was formed between GST-Sec63C and Sec62N-ΔN10-ΔC40, i.e., the truncated Sec62N that lacks the two positively charged oligopeptides (, right panels). The resins were eluted with buffer or nontranslating ribosomes in the same buffer. Subsequently, the resins were eluted with SDS sample buffer, and all samples were analyzed as described above. On elution with buffer, Sec62N and Sec62N-ΔN10-ΔC40 remained bound to the immobilized Sec63C as expected (, lanes 4 and 9). On elution with ribosomes, however, Sec62N eluted together with the ribosomes (lane 3 vs. 5). In contrast, Sec62N-ΔN10-ΔC40 that was unable to bind to ribosomes was not eluted with ribosomes (lane 8 vs. 10). Thus, interaction of Sec62N with Sec63C does not prevent binding of Sec62 to ribosomes, which is consistent with the observed Kd values for the interactions of Sec62N with Sec63C (4.78 nM) and ribosomes (0.13 nM).
Figure 7. Sec62N but not Sec62N-ΔN10-ΔC40 is displaced from Sec63C by ribosomes. GST-Sec63C was immobilized on GSH-Sepharose. Sec62N (lanes 2–5) or Sec62N-ΔN10-ΔC40 (lanes 7–10) were applied to the resin in the presence (more ...)
Mammalian Sec62 Inhibits Translation at the Level of Initiation
ERj1 was shown to be able to bind to ribosomes as well as to inhibit protein synthesis at the level of initiation (Dudek et al., 2005
). Therefore, Sec62N and its derivatives were tested for their ability to inhibit synthesis of firefly luciferase or bovine preprolactin in reticulocyte lysate (, A and B). In this assay, Sec62N and constructs that contained at least a single positively charged oligopeptide were active in inhibiting protein synthesis (although to a varying degree), and the construct with the double deletion (termed Sec62N-ΔN10-ΔC40) was less active (A). Furthermore, yeast Sec62N that lacks similarly charged oligopeptides was inactive (A). We note that a similar inhibitory effect of human Sec62N on the synthesis of luciferase, and preprolactin was observed in the presence of canine pancreatic microsomes (Supplemental Figure 5). A control experiment demonstrated that the translational inhibition activity of Sec62N was specific: the inhibitory effect of Sec62N on translation correlated reciprocally with the ribosome content of the reticulocyte lysate (C).
Figure 8. Inhibition of in vitro protein synthesis by Sec62N and by basic oligopeptides. (A and B) Preprolactin (A) or luciferase (B) were synthesized in reticulocyte lysate in the presence of [35S]methionine. The translation reactions were supplemented with buffer (more ...)
To further substantiate the role of the two highly charged oligopeptides for translational modulation by Sec62N, the synthetic oligopeptides were used in translation (peptides 62-11mer1 and 2; C). Again, the aminoterminal oligopeptide from an invertebrate Sec62 served as negative control (peptide iv62-11mer). Indeed, the aminoterminal peptide from human Sec62N inhibited synthesis of preprolactin as well as luciferase (, D and E).
In addition, Sec62N was tested for its ability to inhibit translation after inhibition of initiation (F). Sec62N did not affect protein synthesis under these conditions. Thus, Sec62N inhibits synthesis of presecretory as well as nonsecretory proteins at the level of initiation.
Mammalian Sec62 Is Protected from Antibody Access by Ribosomes in Permeabilized Cells
Snapp et al. (2004)
established a microscopic method to address the organization of protein translocase in the ER of mammalian cells. The experimental strategy was cell fixation, cell permeabilization, optional ribosome destruction by RNase treatment, and incubation with specific primary and fluorescently labeled secondary antibodies (A). In the subsequent fluorescence microscopy and image analysis, the quantitative data from the RNase-treated cells were compared with the minus RNase control. Positive differential effects in fluorescence intensity were taken as an indication of association of the respective protein with ribosomes. Here, two different cell types (Madin-Darby canine kidney [MDCK] and HeLa) were analyzed with respect to Sec62 (B). The Sec61α and Sec61β subunits of the translocase served as positive controls, the ER-membrane protein calnexin and the ER-lumenal PDI served as negative controls (Snapp et al., 2004
). Although the extents of the differential effects varied between the two cell types, we detected a significant increase in fluorescence intensity after RNase treatment for Sec61α, Sec61β, and Sec62, but not for calnexin and PDI. Similar results were obtained for Cos-7 and HepG2 cells (data not shown). Thus, Sec62 is in the vicinity of ribosomes and, by extrapolation, of Sec61 complexes in the intact ER. This is consistent with our previous findings that Sec61 subunits can be coimmunoprecipitated from microsomal detergent extracts with antibodies against Sec62 and vice versa (Tyedmers et al., 2000
Figure 9. Sec62 is associated with ribosomes at the ER of permeabilized mammalian cells. (A) Experimental strategy. (B) HeLa and MDCK cells were fixed, left untreated or treated with RNase, and labeled with the indicated primary antibodies plus Alexa 555–conjugated (more ...)
To rule out the possibility that the observed epitope protection is due to the ribonucleoprotein particles SRP rather than ribosomes, the α-subunit of the SRP receptor (SRα) was analyzed under identical conditions (C). There was no increase in fluorescence intensity after RNase treatment for SRα in either MDCK or HeLa cells. Thus, the observed epitope protection in the case of Sec61α, Sec61β, and Sec62, was indeed due to ribosomes.
Next we analyzed if the effect of RNase treatment can be mimicked by puromycin treatment of MDCK and HeLa cells, i.e., under conditions where the nascent polypeptides are released from translating ribosomes (). There was no intensity increase detected for any of the proteins after addition of puromycin. This supports the notion that many ribosomes or 60S ribosomal subunits do not leave the ER surface after termination of protein synthesis (Potter et al., 2001
). Furthermore, these data suggested that the RNase experiments visualized translating as well as nontranslating ribosomes.
Figure 10. Sec62 is associated with nontranslating ribosomes at the ER of permeabilized mammalian cells. HeLa and MDCK cells were incubated in the absence or presence of the indicated concentrations of puromycin for 1 h at 37°C. Then the cells were fixed (more ...)