ER retention may play a role in sorting of the nuclear pore membrane protein POM121

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Experimental Cell Research 284 (2003) 173–184

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ER retention may play a role in sorting of the nuclear pore membrane protein POM121 G. Imreh,a,b D. Maksel,a J.B. de Monvel,c L. Brande´n,d and Einar Hallberga,* a So¨derto¨rns Ho¨gskola (University College), S-141 89 Huddinge, Sweden Department of Biochemistry and Biophysics, Stockholm University, 106 91 Stockholm, Sweden c Institute for Hearing and Communication Research M9:01-ENT, Karolinska Hospital S-171 76 Stockholm d Center for Biotechnology, NOVUM, Karolinska Institute, S-141 57 Huddinge, Sweden b

Received 27 June 2002, revised version received 11 September 2002

Abstract Integral membrane proteins of the nuclear envelope (NE) are synthesized on the rough endoplasmic reticulum (ER) and following free diffusion in the continuous ER/NE membrane system are targeted to their proper destinations due to interactions of specific domains with other components of the NE. By studying the intracellular distribution and dynamics of a deletion mutant of an integral membrane protein of the nuclear pores, POM121, which lacks the pore-targeting domain, we investigated if ER retention plays a role in sorting of integral membrane proteins to the nuclear envelope. A nascent membrane protein lacking sorting determinants is believed to diffuse laterally in the continuous ER/NE lipid bilayer and expected to follow vesicular traffic to the plasma membrane. The GFP-tagged deletion mutant, POM1211–129-GFP, specifically distributed within the ER membrane, but was completely absent from the Golgi compartment and the plasma membrane. Experiments using fluorescence recovery after photobleaching (FRAP) and fluorescence loss in photobleaching (FLIP) demonstrated that despite having very high mobility within the whole ER network (D ⫽ 0.41 ⫾ 0.11 ␮m2/s) POM1211–129-GFP was unable to exit the ER. It was also not detected in post-ER compartments of cells incubated at 15°C. Taken together, these experiments show that amino acids 1–129 of POM121 are able to retain GFP in the ER membrane and suggest that this retention occurs by a direct mechanism rather than by a retrieval mechanism. Our data suggest that ER retention might be important for sorting of POM121 to the nuclear pores. © 2003 Elsevier Science (USA). All rights reserved. Keywords: Nuclear pores; Endoplasmic reticulum; Green fluorescent protein; Integral membrane protein; Protein trafficking; FRAP; FLIP

Introduction Protein sorting to the endoplasmic reticulum (ER) and post-ER compartments occurs at several levels and requires multiple sorting signals and multiple sorting events [1–3]. After synthesis at the RER, most proteins are selectively targeted to their proper destination, such as the lysosomes, Golgi compartments, or the cellular exterior. Soluble and membrane-bound ER resident proteins are either directly retained in the ER or retrieved from post-ER compartments in a receptor-mediated process. Retention of soluble lumenal ER proteins is mediated by a short KDEL motif at the * Corresponding author. Section for Natural Sciences, So¨dertorns Ho¨gskola, S-141 89 Huddinge, Sweden. Fax: ⫹46-8-6084510. E-mail address: [email protected] (E. Hallberg).

C-terminal end which functions as a transport signal for retrieval from the pre-Golgi compartments to ER by the KDEL cargo receptor [4]. ER resident integral membrane proteins are synthesized at the RER and then either exported by vesicular traffic and retrieved back from post-ER compartments or directly retained in the ER. The retrieval of integral membrane proteins from the post-ER organelles is directed by cytoplasmic KKXX or KXKXX motif located at the C-terminus of type I membrane proteins [5– 8] or XXRR motif located at the N-terminus of type II proteins [9]. In addition, a number of membrane proteins that do not contain any of these signals are retrieved back to the ER by other mechanisms [10]. Finally, direct retention of integral membrane proteins in the ER has also been described [11–17], although the mechanism of such retention has not been elucidated.

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The nuclear envelope surrounding the nucleus can be regarded as a continuous extension of ER that has specific functions and is occupied by specific nuclear membrane proteins. It consists of two concentric membranes, the outer nuclear membrane (ONM) that is continuous with the ER and the inner nuclear membrane (INM). The ONM and INM are fused together at the thousands of nuclear pores perforating the NE [for review see 18,19]. Integral membrane proteins that are destined for nuclear membranes are also synthesized on the RER but their sorting has been subjected to only a few studies. According to the general view, after synthesis on the RER and incorporation into the membrane, proteins destined for the nuclear pores or the inner nuclear membrane are able to diffuse laterally in the continuous ER/nuclear membrane system before becoming immobilized by binding to other components at their proper location. The binding domain of the protein is thus believed to determine its final destination in the cell [20 –26]. However, one should keep in mind that the nuclear membrane is reversibly disassembled and regenerated during every cell division. Nuclear membrane proteins diffuse out into ER in pro/metaphase and must be sorted back to the nuclear pore membrane or inner nuclear membrane after chromosome segregation, [23,27,28]. Therefore, prior to reaching the nuclear membrane after synthesis or being retracted after mitosis, in the absence of ER retention signals these proteins could potentially enter vesicles budding off from the ER and thus follow default vesicular traffic to the plasma membrane, which would make sorting of these proteins to the nuclear membrane inefficient. A mechanism for retaining nuclear membrane proteins in the ER would certainly be beneficial for sorting to the nuclear membrane, but to our knowledge no such mechanism has so far been described or reported. POM121 is one of two known integral membrane proteins of the nuclear pores, which are thought to take part in nuclear pore formation [29,30]. It has a short amino-terminal tail facing the perinuclear space, a single transmembrane segment, and a large carboxy-terminal domain adjoining the nuclear pore complex [31]. POM121 is also located at the pores in annulate lamellae (AL) [32], which are arrays of stacked membranes present in the cytoplasm of some cell types and believed to function as storage compartment for excess nuclear pore proteins [33]. We have shown previously that the amino acids 1–129 were sufficient for targeting GFP to the ER whereas the amino acids 129 – 618 in the carboxy-terminal portion of POM121 were necessary for targeting to the nuclear pores [25], suggesting that this latter domain is able to interact with other NPC proteins. Here we investigate the intracellular distribution and dynamics of a GFP-tagged fragment of POM121, POM1211–129-GFP, that lacks the nuclear pore-targeting domain. A possible role for ER retention in inward trafficking of POM121 and other integral membrane proteins of the nuclear membrane is discussed.

Materials and methods DNA constructs In order to construct hybrid DNA encoding chimerical POM1211–129-GFP fusion protein, the 5⬘-end of cDNA encoding a GFP (F64L, S65T) double mutant [34] was fused to the 3⬘-end of cDNA encoding POM1211–129 and then incorporated into an eukaryotic expression vector as described [25]. The plasmid encoding KDELR-GFP [35] was a generous gift from Dr. J. Lipincot-Schwartz. Cell culture, transfection, and treatment COS-7 (African green monkey kidney) and BHK (Syrian hamster kidney) cells were grown in Dulbecco’s minimal essential medium (DMEM) supplemented with 10% FCS and 50 ␮g/mL gentamicin. The cells were cultured at 37°C on glass coverslips and transiently transfected using the calcium phosphate method [36] or FuGENE 6 transfection reagent (Roche Molecular Biochemicals, Stockholm, Sweden). At different times posttransfection the cells were prepared for immunofluorescence, Western blotting, or timelapse studies. To verify membrane topology, we used digitonin at a concentration of 40 ␮g/mL and 0.5% TX-100 as described [31]. To inhibit protein synthesis, COS-7 cells transiently expressing POM1211–129-GFP were treated with cycloheximide at a concentration of 100 ␮g/mL [37] and prepared for Western blotting. Subcellular localization of the chimerical GFP-tagged protein by fluorescence microscopy was analyzed in cells transfected for 36 h, incubated at 15°C for 2.5 h, and then fixed and prepared for immunofluorescence (all chemicals were purchased from Sigma-Aldrich AB, Stockholm, Sweden). Antibodies Polyclonal anti-POM121 (rat) antibodies were raised against the COOH-terminal (amino acids 660 – 800) of rat POM121 [38], anti-GFP monoclonal antibodies (No. 8362) were purchased from Clontech Laboratories Inc. (Palo Alto, CA), and monoclonal anti-c-Myc antibodies (9E10) were from Sigma-Aldrich AB (Stockholm, Sweden). As a marker for the ER compartment we used affinity-purified rabbit polyclonal antibodies raised against a synthetic peptide corresponding to amino acids 556 –573 of the endoplasmic reticulum protein calnexin [39] (kindly donated by Dr. R. Pettersson). For identification of endogenous p58 protein, we used mouse monoclonal anti-p58 antibodies (BioSite, Stockholm, Sweden). Tetramethylrhodamine isothiocyanate (TRITC)-conjugated goat anti-mouse IgG, TRITC-conjugated anti-rabbit IgG (from Jackson ImmunoResearch Laboratories Inc., West Grove, PA), and Alexa Fluor 568 goat anti-mouse IgG (Molecular Probes, Leiden, Netherlands)

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were used as secondary antibodies. To visualize the plasma membrane we used rhodamine-labeled WGA. Immunofluorescence and Western blotting Cells grown on coverslips were transiently transfected as described above and at different times posttransfection prepared for immunofluorescence and fluorescence microscopy as described [25,31]. For Western blotting, SDS-PAGEseparated proteins were transferred onto nitrocellulose membranes, which were blotted against the primary polyclonal anti-POM121, monoclonal anti-GFP or anti-c-Myc antibodies, followed by HRP-coupled secondary antibodies and ECL detection (Amersham Pharmacia Biotech, Uppsala, Sweden). Western blots were analyzed using the luminescent image analyzer, LAS1000plus system, and quantified using the ImageGauge 3.1 software (Fuji Photo Film Co., Ltd., Tokyo, Japan). Fluorescence microscopy and image processing The samples prepared for immunofluorescence were analyzed with a confocal laser scanning microscope (Model Leica TCS-SP Stockholm, Sweden) equipped with Ar-Kr lasers and a 63x 1.4 oil immersion objective. The intensity of the laser was set at the lowest point possible to avoid photobleaching. The images acquired were processed using Adobe Photoshop 5.0 software (Adobe systems Inc., CA). FRAP (fluorescence recovery after photobleaching) experiments were performed on a temperature-controlled stage of a Leica TCS-SP confocal microscope equipped with a 100⫻ 1.4 NA objective using a 488-nm laser excitation line of a 20 mW Ar laser. A defined region was bleached at full laser power for 2.5 s and recovery of fluorescence monitored by scanning the whole cell at low laser power. Recovery curves were generated by comparing the intensity ratio in regions of interest inside and outside the bleached area before bleach and during recovery. The intensities were normalized to correct for total loss of fluorescence due to overall photobleaching as described previously [40]. For D value calculations we followed the approach described in [41]. In FLIP (fluorescence loss in photobleaching) experiments a defined region within the cell was repeatedly bleached at full laser power for 1 s followed by image acquisition at 1-s intervals.

Results Intracellular distribution and topology of POM1211–129-GFP fusion protein We have studied the intracellular distribution of POM1211–129-GFP in transiently transfected tissue culture cells. POM1211–129-GFP is a recombinant fragment of the pore membrane protein POM121 fused to GFP; containing

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POM121’s luminally exposed 28-residue N-terminal tail, the 44-residue hydrophobic transmembrane domain, and the first 57 residues of the cytoplasmically exposed C-terminal domain. The fragment is able to direct GFP to the ER, but lacks signal(s) for targeting to the NPC and was previously shown to locate in the ER [25]. Examination at the level of resolution of confocal laser scanning microscopy (CLSM) in this study showed that POM1211–129-GFP distributed within the ER and perfectly colocalized with immunostaining using antibodies specific for the ER resident protein calnexin (Figs. 1a– c). Identical distribution patterns were observed in other cell lines (RC-37, BHK, Rat-2) overexpressing POM1211–129-GFP (not shown). Next we wanted to confirm that neither fusion to GFP nor truncation of 1070 amino acids from the C-terminal portion of POM121 (bitopic type I integral membrane protein) affected the membrane topology. The accessibility of the GFP moiety of POM1211–129-GFP for anti-GFP antibodies was inspected by immunofluorescence microscopy in transfected cells treated with digitonin in order to selectively permeabilize the plasma membrane without perturbing other membranes, or Triton X-100 in order to permeabilize all membranes [31]. In both digitonin-treated cells (Figs. 1d–f) and Triton X-100-treated cells (Figs. 1g–i) the anti-GFP antibody gave rise to a typical ER staining pattern identical to the distribution pattern of GFP fluorescence, indicating that the Cterminal GFP domain of POM1211–129-GFP was cytoplasmically exposed, and demonstrating that topology of POM121 was preserved in POM1211–129-GFP. No GFP fluorescence was detected in the plasma membrane (Figs. 1j–l). Finally, the distribution of POM1211–129-GFP did not colocalize with immunostaining against Golgi resident protein mannosidase II in BHK cells (which were used in this experiment due to the unavailability of antibodies against human mannosidase II, Figs. 1m– o) and was also distinct from the distribution of the ERGIC marker p58 (see Fig. 5a). Taken together our results indicate that POM1211–129GFP becomes correctly inserted in the ER membrane and appears to be retained in the ER rather than to follow default vesicular traffic to post-ER compartments and the plasma membrane. Turnover of POM1211–129-GFP in COS cells The apparently restricted distribution of POM1211–129GFP in the ER could in theory result from a combined high expression in COS-7 cells and rapid degradation due to improper folding or maturation under such conditions. The fact that GFP becomes fluorescent argues against improper folding, but in order to rule out the possibility of rapid synthesis and degradation we compared the turnover of POM1211–129-GFP with full-length rat POM121 in transfected COS-7 cells after inhibition of protein synthesis with cycloheximide (Fig. 2a). Both POM1211–129-GFP and fulllength POM121 were degraded only slowly and displayed a 38 and 50% reduction, respectively, after 9 h of cyclohex-

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even slower degradation of POM1211–129-GFP rules out the possibility that its apparently restricted location in the ER results from rapid synthesis and degradation and supports the idea that POM1211–129-GFP is indeed retained in the ER. Dynamic properties of POM1211–129-GFP in living cells

Fig. 2. Turnover of overexpressed full-length POM121 and POM1211–129GFP. COS-7 cells were transfected either with cDNA encoding full-length rat POM121 or POM1211–129-GFP. At 36 h posttransfection the cells were treated with 100 ␮g/mL cycloheximide. (A) Total proteins of transfected cell cultures incubated in the absence (⫺) or presence (⫹) of cycloheximide for the indicated length of time were separated by SDS-PAGE, transferred to nitrocellulose membrane, and immunoblotted using antibodies against POM121 for detection of overexpressed rat POM121 and anti-GFP antibodies for detection of overexpressed POM1211–129-GFP, respectively. Endogenous c-Myc was analyzed using antibodies specific for c-Myc. (B) Quantification of Western blots. The intensities of the bands on the Western blot in A were expressed as percentages of control and plotted versus number of hours of cycloheximide treatment.

imide treatment. In contrast, c-Myc, which has a high reported turnover [42] and thus was used to control that protein synthesis inhibition was effective, was degraded at a much higher rate. The slow degradation of overexpressed full-length POM121 is consistent with a recent study setting the half-time for dynamic turnover of POM121-GFP3 located in the nuclear pores of NRK cells to 20 h [28]. The

To investigate how POM1211–129-GFP is retained in the ER we examined its dynamic properties in live cells. Retention in a cellular compartment can in principle be caused by aggregation with itself or strong interaction with other components in the ER, both of which would restrict the mobility of POM1211–129-GFP. However, FRAP experiments showed that recovery of fluorescence in a bleached area of the ER was almost complete in less than 1 min, with a half-time of ⬃8 s (Fig. 3). Quantification of FRAP data showed that POM1211–129-GFP was 95% mobile and had a large diffusion coefficient (D ⫽ 0.41 ⫾ 0.11 ␮m2/s, n ⫽ 7), comparable to that of other GFP-tagged proteins residing in the ER [35,43,44]. The obtained D value is also consistent with the diffusion constant recently reported for fulllength POM121-GFP3 freely diffusing in the ER of metaphase cells (D ⫽ 0.25 ␮m2/s; [28]) taking into account the smaller size of the cytoplasmically exposed portion of POM1211–129-GFP as compared to POM121-GFP3. The large mobility of POM1211–129-GFP strongly argues against retention resulting from an inability to exit ER due to aggregation or a strong interaction with ER components. A number of ER resident proteins undergo a continuous receptor-mediated vesicular cycling between ER and post-ER compartments such as ER-Golgi intermediate compartment (ERGIC) or cis-Golgi. FLIP is a sensitive and accurate technique for detection of dynamic cycling between cellular compartments. Continuous cycling is manifested as fluorescence remaining in an acceptor compartment after complete quenching of the donor compartment by repeated bleaching [45]. After repetitive photobleaching of POM1211–129-GFP in a selected area of the ER uniform loss of fluorescence from the entire ER was observed with no remaining patches of fluorescence in post-ER compartments (Fig. 4A, upper panels), strongly suggesting that POM1211–129-GFP does not undergo cycling between the ER and post-ER compartments. In contrast, in cells expressing KDEL receptor fused to GFP (KDELR-GFP), which

Fig. 1. POM1211–129-GFP is specifically distributed in the endoplasmic reticulum. COS-7 and BHK cells transiently transfected with cDNA encoding POM1211–121-GFP were examined by confocal laser scanning fluorescence and immunofluorescence microscopy. In COS-7 cells the GFP fluorescence distributed in the nuclear rim and in a reticular pattern in the cytoplasm (a, d, g, j) as did the immunostaining of the endogenous ER resident protein calnexin, (b). The GFP fluorescence and the anticalnexin staining perfectly colocalized when the corresponding images were superimposed (c). Transfected COS-7 cells were treated either with digitonin (d–f) or Triton X-100 (g–i) before fixation and immunostaining using antibodies specific for GFP. Anti-GFP immunostaining visualized in the TRITC channel (e and h) colocalized perfectly with the GFP fluorescence of digitonin-treated cells (d) and Triton X-100-treated cells (g) when superimposed (f, i). Fixed but nonpermeabilized COS-7 cells expressing POM1211–121-GFP were stained using rhodamine-labeled WGA (j–l). WGA gave rise to an intensive staining of the cell surface (k). The GFP fluorescence (j) and plasma membrane staining (k) did not colocalize at all when superimposed (f). BHK cells transfected with POM1211–121-GFP were immunostained using antibodies specific for rat mannosidase II (m– o). The distribution pattern of the GFP fluorescence (m) was distinct from the distribution of mannosidase II (n) in the transfected cell. Bars, 10 ␮m.

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Fig. 3. FRAP analysis of diffusional mobility of POM1211–129-GFP in the ER membrane of living cells. COS-7 cells were transfected with cDNA encoding POM1211–129-GFP. (A). A single spot in the ER of a cell expressing POM1211–121-GFP indicated by a white circle was bleached at full laser power followed by sequential acquisition of images at low laser power. Note the recovery of fluorescence in the bleach spot at 8 and 57 s. (B) Quantitative FRAP. Seven different COS-7 cells expressing POM1211–129-GFP were spot bleached as in A. Immediately after bleaching 15 images were acquired in 2-s intervals followed by further acquisitions every 5 s. Percentages of recovery of the fluorescence intensity relative to the prebleach value in each of the bleached spots are plotted versus time.

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Fig. 4. FLIP analysis of distribution of POM1211–129-GFP and KDELR-GFP in cellular membranes. (A) COS-7 cells expressing POM1211–129-GFP (upper panels) or KDELR-GFP (lower panels) were repeatedly bleached for 1 s at high laser power in a single spot within the ER (white circles). Between bleaches, the entire field of view was imaged at low laser power. After repeated photobleaching, in cells expressing POM1211–129-GFP, the fluorescence disappeared homogenously from the entire ER network without leaving any fluorescing structures (upper panels), in contrast to cells expressing KDELR-GFP in which residual fluorescent structures remained (lower panels). (B) Intensities of GFP fluorescence as measured outside of the bleached regions in cells depicted in A: (Œ) ER of the cell expressing POM1211–129-GFP, upper panel; (■) ER and (}) Golgi compartment of the cell expressing KDELR-GFP, lower panel.

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cycles rapidly between ER and post-ER compartments, repetitive photobleaching removed the fluorescence from the ER but left significant amounts of fluorescence in post-ER compartments (Fig. 4, lower panels). This is in agreement with previous observations [35,44] that recycling of KDELR-GFP between ER and post-ER compartments is slower than lateral diffusion within the membrane of ER. We also studied distribution of POM1211–129-GFP in COS-7 cells incubated at low temperature to allow for accumulation of proteins exiting ER in the post-ER compartment. It has been shown previously that at reduced temperatures protein movement is arrested and while exit of protein cargo from ER is still allowed, at 15°C they accumulate in the ERGIC, whereas at 20°C accumulation occurs within the Golgi complex [46,47]. Such cold-induced traffic blocks were efficiently used to trap proteins exported from ER by immobilizing the ERGIC vesicles near ER [48]. To visualize ERGIC in COS-7 cells we used antibody against protein p58 [6,49], which is type I membrane protein recycling constitutively between ER, ERGIC, and cis-Golgi [50,51, and references therein]. Peripheral elements that stained positively for p58 were inspected for fluorescence in cells incubated at 37°C or shifted to 15°C for 2.5 h. No colocalization of GFP fluorescence with the anti-p58 staining was detected and the GFP fluorescence remained localized in the ER in both untreated and cold treated cells (Fig. 5). The pixel intensity graphs show different profiles for the green and red channels, indicating that POM1211–129-GFP and ERGIC marker, p58, distribute in spatially separated compartments in both the untreated and the cold-treated cell. These results are thus in support of the data from dynamic studies and taken together they strongly suggest that POM1211–129-GFP is directly retained in the ER rather than transported to post-ER compartments and then retrieved back to the ER.

Discussion We have investigated the intracellular distribution and dynamics of POM1211–129-GFP, a GFP-tagged fragment of the nuclear pore membrane protein POM121, which is synthesized on the RER but lacks signals for sorting to the nuclear pores. Using confocal laser scanning microscopy we have shown that POM1211–129-GFP exclusively distributes in the ER-nuclear membrane system and is absent from the post-ER compartments or plasma membrane, indicating

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that it is retained in the ER. Photobleaching experiments demonstrated that although POM1211–129-GFP diffuses freely within the ER membrane it does not exit ER, which suggests that the first 129 amino acid residues of POM121 are able to mediate direct retention in the ER. What keeps POM1211–129-GFP in the ER? POM121 does not contain any of the known signals for ER retention within its first 129 amino acids, although the possible presence of a yet unidentified retention signal cannot be excluded. In an ongoing study, to exactly define the ER retention determinants in POM121, we have found that the hydrophobic domain (residues 29 –73) fused to GFP distributed as POM1211–129-GFP (unpublished data), suggesting that the TMD alone is sufficient to mediate ER retention. It has been proposed that length of a hydrophobic TMDs is an important discriminatory factor in distinguishing proteins localized to the membrane of the Golgi apparatus or PM [52,53]. On the other hand, there is no obvious difference in the lengths or sequences of TMDs of ER and Golgi membrane proteins, suggesting that other properties within these sequences are responsible for the selective localization of proteins in either organelle. Thus, in additional to length, both the distribution of hydrophobicity and the presence of polar residues within the TMD play important roles as sorting determinants [54 –56]. The picture is further complicated by contribution from flanking regions [54,57–59] and in some cases from more distant multiple domains [14,50,60 – 62]. Finally, ER retention could result from incorporation of membrane proteins into large immobile networks [63,64] that fail to enter transport vesicles or are formed in the regions of the ER not involved in transport vesicle formation. Nevertheless, this mechanism fails to explain the ER retention of proteins that have been shown to have high lateral mobility [43,65]. Our dynamic investigation showed that virtually the whole population of POM1211–129-GFP molecules was able to diffuse very rapidly in the lateral plane of the ER membrane. This suggests that POM1211–129-GFP does not form oligomeric assemblies, as aggregates too large to be packaged in transport vesicles are unlikely to be dynamic enough not to influence protein mobility. However, the unusual 44 amino acid residue long hydrophobic domain of POM121 [31] could potentially contain the information necessary for retention in the ER. A TMS inserted obliquely in the membrane may affect the effi-

Fig. 5. Distribution of POM1211–121-GFP in cold-treated cells. COS-7 cells were transiently transfected with cDNA encoding POM1211–121-GFP. At 36 h posttransfection, cells were submitted to cold treatment for 2.5 h at 15°C and then prepared for immunofluorescence. The GFP fluorescence (a, d) and the immunostaining with antibodies specific for an ERGIC marker p58 (b, e) are shown separately or superimposed (c, f). Note that in the control as well as the cold-treated cell, the GFP fluorescence was retained in the reticular network of the ER, whereas the p58 protein was found confined to a number of small vesicles (arrow heads) and a bigger structure most likely representing the cis-Golgi compartment. Analysis of pixel intensity in the green and red channel along the white line in the central region of the cell incubated at 37°C (g) or in the cell incubated at 15°C (h) shows that the distribution profiles of GFP fluorescence and anti-p58 immunostaining are different. The numbered sharp peaks of red fluorescence correspond to the vesicles shown by respective arrowheads in (b) and (e). Bar, 10 ␮m.

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ciency of packaging into transport vesicles. There is correlation between the predicted tilt of secretory signals and the efficiency in mediating secretion [66], as well as between tilted TMS of some viral proteins and membrane fusion [67]. Another possibility could be that the proline 52 in the middle of the hydrophobic domain may break the TMS of POM121 resulting in 2 perpendicular ␣-helical hydrophobic domains that would potentially influence the efficiency of packaging and vesicle formation. Do nuclear membrane proteins need ER retention? Several lines of evidence suggest that pore membrane protein POM121 is involved in formation of the nuclear and annulate membranes: it is present in nuclear and annulate lamellae pores and massive overexpression of POM121 results in de novo formation of AL-like structures in COS cells normally lacking AL [32]. In addition, POM121 is one of the earliest proteins to be recruited to the reforming nuclear membrane in late anaphase [28,68]. Formation of nuclear pores requires intralumenal transcisternal membrane fusion. It is believed that this fusion is mediated by oligomerization of pore membrane proteins [29] and stabilized by binding of peripheral pore proteins, which build up the NPC architecture. Thus, it is possible that POM121 has an inherent ability to oligomerize at specific nucleation sites for pore formation. In any case retention of pore membrane proteins in the ER seems to be important in order to achieve necessary time and concentration for the transcisternal membrane fusion event during formation of nuclear or AL pores. A survey of the literature indicates that ER retention might also be important for other nuclear membrane proteins. Studies of proteins of the inner nuclear membrane and pore membrane, which were unable to target to their proper location due to excessive overexpression or deletion of their targeting/immobilization domains, showed that with the exception of emerin that was present in the plasma membrane [24], these proteins, i.e., LBR [20,23,69], LAP2 [22], MAN1 [70], and nurim [71], were reported to distribute only in the ER. Furthermore, the presence of nuclear membrane proteins on the cell surface could have undesirable consequences for the cell’s well-being as specific autoantibodies against nuclear envelope proteins, including constituents of nuclear pore complexes, have been detected in patients with rheumatic diseases, primary biliary cirrhosis, and autoimmune hepatitis [72–74]. Thus, the available data suggest that ER retention may commonly play a role in sorting of nuclear membrane proteins. Together these reports and our findings raise a number of interesting questions concerning the role of ER retention in inward trafficking of nuclear membrane proteins. In general, even if targeting of nuclear membrane proteins to the inner nuclear membrane and pore membrane could be explained by a diffusion/immobilization mechanism alone, ER retention would clearly facilitate sorting of nascent proteins by pre-

venting their escape via default vesicular transport. Experiments to elucidate the mechanism underlying the ER retention of POM121 are currently underway.

Acknowledgments The authors thank Drs. A. Andersson and R. Pettersson (LICR, Karolinska Institute) for antibodies against calnexin. This work was supported by the Swedish Research Council, Carl Tryggers Stiftelse, Mang. Bengvalls Stiftelse, and Stiftelsen Wenner-Grenska Samfundet.

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