Expression of Sec61β in wild type and sec61βP1germline clones
The sec61βP1allele has been reported to be a probable loss-of-function allele [5]. In order to characterise the allele in greater detail we generated an antibody against the N-terminus of Sec61β. This antibody was used for western blot analysis using protein extracts from wild type ovaries and ovaries derived from female germline homozygous for sec61βP1(Fig. 1A). The amount of Sec61β protein is significantly reduced in ovaries derived from homozygous mutant clones as compared to the wild type ovaries. The small amount of Sec61β protein observed in sec61βP1homozygous clones most likely represents Sec61β in follicle cells. Follicle cells do not originate from the germ cell lineage and should retain normal amounts of Sec61β [11].
Localization of the Gurken mRNA is unaffected in the germline clones of Sec61β
The germline clones of the sec61βP1allele generated embryos with fused dorsal appendages indicating perturbations in the dorsal-ventral axis [5]. The observed phenotype is strongly reminiscent of the dorsal-ventral axis defect found in the Gurken mutations. Asymmetric localization of the Gurken mRNA at the anterior-dorsal end of the ooycte is the first step towards establishment of the dorsal-ventral axis [12]. It has been proposed that the Gurken mRNA is closely associated with the ER and an ER protein could anchor the mRNA at the anterior-dorsal end of the oocyte [13].
We therefore examined the localization of Gurken mRNA in the stage 10 egg chambers by in situ hybridisation. In wild type oocytes Gurken mRNA localizes to the anterior-dorsal end of the oocyte in close proximity to the oocyte nucleus as indicated in the cartoon (Fig. 1B and 1C). In sec61β mutant oocytes Gurken mRNA similarly localizes to the anterior-dorsal end of the oocyte (Fig 1C and 1D). Thus, the sec61βP1germline clones show no apparent difference in the localization of Gurken mRNA in oocytes.
Germline clones of sec61βP1allele show reduced levels of Gurken protein at the plasma membrane of the oocyte
Since the mRNA localization of Gurken was not affected by lack of Sec61β, we investigated whether lack of Sec61β affects the translation of Gurken mRNA or events in the transport of Gurken protein to the plasma membrane.
We performed immuno-fluorescence analysis on wild type egg chambers or germline clones of sec61βP1using a monoclonal antibody generated using the N-terminal extra-cellular domain of the Gurken protein (Developmental Studies Hybridoma Bank, [14]). The egg chambers were also stained with phalloidin, which binds actin localized below the plasma membrane and serves to delineate the plasma membrane. In stage 10 wild type oocytes Gurken protein localizes at the anterior-dorsal part of the oocyte in close proximity to the oocyte nucleus in precisely the same region as the mRNA (Fig. 2A). The mutant stage 10 oocytes also stains positive for Gurken, with the protein localized in the anterior-dorsal end of the oocyte (Fig. 2B). Upon analysis of the anterior-dorsal part of the oocytes at higher magnification we observed Gurken at two different locations in the oocyte, in punctuate structures in the cytoplasm between the plasma membrane and the nucleus and at the plasma membrane at the anterior-dorsal end of the oocyte, co-localizing with actin, directly opposite the follicle cells or the nurse cells (Fig. 2C). In the oocytes derived from the germline clones of sec61βP1the cytoplasmic pool of Gurken protein was observed at the anterior-dorsal part of the oocyte in punctuate structures similar to the wild type oocytes but the Gurken protein amount at the plasma membrane was drastically reduced (Fig. 2D). Reduction in Gurken amount occurs in the part of the plasma membrane that is in direct apposition to the follicle cells and the nurse cells (Fig. 2D). To rule out the possibility that the observed changes in Gurken localization observed were due to differences in the focal plane in view, we analysed series of optical sections from different depths of the wild type oocytes (Fig. 2ES1-S4) and the mutant oocyte (Fig. 2FS1-S4). In all cases Gurken protein is excluded specifically from the plasma membrane.
We also observed staining for Gurken protein in distinct speckles inside the follicle cells at the anterior-dorsal end of the oocytes (Fig. 2G). These speckles most likely represent the protein that has been internalized by the follicle cells. In sec61βP1germline clones on the other hand, Gurken staining in the follicle cells is rarely observed (Fig. 2H). Taken together, these results demonstrate that egg chambers from the sec61βP1germline clones have reduced levels of Gurken protein at the plasma membrane of the oocyte and in the surrounding follicle cells.
Gurken protein is also mis-localized during early oogenesis
Gurken protein signals to the EGF receptor on the follicle cells at two different stages of oogenesis. During late oogenesis (stage 10–11) Gurken signals to follicle cells at the anterior-dorsal end of the oocyte. During earlier stages of oogenesis (stages 6–9) the oocyte nucleus and the Gurken mRNA are localized at the posterior part of the oocyte with Gurken protein signalling to the posterior follicle cells [12]. In order to investigate the localization of Gurken during early oogenesis we co-stained egg chambers during stage 6–9 of oogenesis with the anti-Gurken antibody and with phalloidin. The Gurken protein in the wild type egg chambers is localized in punctuate cytoplasmic structures towards the posterior part of the oocyte (Fig 3A and 3B). We also observe Gurken staining in the posterior follicle cells. In the oocytes derived from sec61βP1germline clones we observe Gurken in the oocyte cytoplasm similar to wild type oocytes (Fig 3C and 3D) but not in the posterior follicle cells (Fig 3D). Thus, the Gurken trafficking defect is also observed during early stages of oogenesis.
The general structure and function of ER remains unaffectedin sec61β mutant oocytes
Gurken is a type I membrane protein and the presence of the signal sequence suggests that the protein is most likely co-translationally translocated into the ER and transported along the secretory pathway to reach the plasma membrane. To investigate if the mislocalization of Gurken was due to a general impairment in structure and function of ER, we co-stained wild type and sec61β mutant egg chambers with the Gurken antibody and an antibody raised against the Boca protein that has been previously characterised as an ER resident protein in oocytes [15]. In wild type egg chambers during stage 9–10 of oogenesis we observe a diffused staining for Boca in a region below the plasma membrane throughout the oocyte (Figure 4A). Oocytes derived from the sec61βP1germline clones show a very similar staining, suggesting that the overall organization of the ER remains largely unaffected by lack of Sec61β(Fig. 4C). Localization of Gurken in the wild type and the sec61β mutant oocytes is as observed previously (Fig 4B,D, and Fig 2).
The Sec61 translocon translocates a variety of secretory and membrane proteins into the ER. The reduction in the Gurken amounts at the plasma membrane in the germline clones of sec61βP1could be due to a general defect in the plasma membrane trafficking. In order to investigate this possibility we examined the localization of another plasma membrane protein of the oocyte, Yolkless (Yl). Yl is a type I membrane protein expressed by the oocytes for the uptake of vitellogenins and yolk proteins during oogenesis [16]. We co-stained wild type and mutant oocytes for Yolkless (Yl) and actin. Upon staining we observed that Yl is localized to the oocyte plasma membrane in both the wild type egg chambers (Fig.4E–G) and sec61βP1homozygous clones (Fig.4H–J) during stage 9–10 of oogenesis, thus suggesting that Sec61β does not cause a general defect in protein trafficking.
Sec61β is not required for ER translocation
Data obtained from immunofluorescence analysis in oocytes so far suggest that Sec61β was involved in the transport of the Gurken protein to the plasma membrane, however it was not clear which step of the transport process is affected. We wanted to further characterise this defect and to test the actual process of ER translocation using HeLa cells. Previous studies have also utilized the mammalian cell cultures system for investigating trafficking of EGFR ligands. Gurken and other EGF ligands such as Spitz and Keren can be expressed and secreted in mammalian cells. The process requires Star for ER exit and Rho for proteolytic processing, thus in part recapitulating the trafficking of EGFR ligands as it occurs in the fly system [17, 18]. We used this system to examine if changes in the amount of Sec61β could affect the translocation of Gurken into the ER.
Gurken was transfected into HeLa cells, either alone or co-transfected with the regulatory proteins, Star and Rho. On western blot analysis of the whole cell lysate, Gurken is seen to migrate as a 45–47 KDa protein (Fig. 5A). Upon co-transfection with Myc-tagged Star the position of the band is unchanged. When HA-tagged Rho is transfected, Gurken shows faster migration, which most likely represents the intra-membrane cleaved form of Gurken. Gurken is glycosylated as inferred from its sensitivity to Endo-glycosidase H, EndoH (Fig. 5A, right panel). Sensitivity to EndoH is retained even when Star and Rho are transfected along with Gurken indicating retention in the ER. Based on this data we think in this cell type Star does not cause an extensive Gurken export from the ER and Rho is able to process the Gurken within the ER. Expression of Star and Rhomboid was confirmed by western blot analysis using the anti-Myc and anti-HA antibody respectively (data not shown).
In order to investigate Gurken translocation into the ER in the absence of Sec61β, a plasmid was transfected into HeLa cells that was designed to generate double stranded RNA oligo against Sec61β mRNA. As a control, a plasmid designed to generate a scrambled double stranded RNA oligo was also transfected. Three days after the initial transfection the cells were re-transfected with the Sec61β siRNA plasmid or the control siRNA plasmid, together with the expression plasmids for Gurken, Star and Rho. One day after the second round of transfections (and four days after the initial round of transfection) Sec61β protein is no longer detected by western blotting in cells transfected with the Sec61β siRNA plasmid, whereas cells transfected with the control plasmid retain normal level of Sec61β protein (Fig. 5B). Expression of Star and Rho is confirmed by western blot analysis using the anti-Myc and anti-HA antibodies respectively (Fig 5C).
ER translocation of Gurken was investigated as follows: a pulse analysis using 35S-methionine was performed and the cells were harvested for immuno-precipitation and western blotting. The actual process of translocation and availability of Gurken for post-translocational processing was determined by the extent of glycosylated protein. Correctly translocated Gurken would be glycosylated in the ER, on the other hand, conditions affecting translocation or downstream events would result in a species of Gurken that is unavailable for glycosylation.
The cell lysates after 35S-methionine pulse were probed with antibody against the Gurken protein (Fig 5D). Gurken is visible in the processed, faster migrating form in control cell lysates and lysates with depeleted Sec61β protein. Gurken is EndoH sensitive in cells with normal and reduced Sec61β protein, hence glycosylated and present in the ER (Fig 5D). Guken protein was immuno-precipitated from control and Sec61β depleted cells; half of the immuno-precipitated sample was treated with EndoH and the other half was left untreated, both the samples were applied to SDS-PAGE. We observe that in lanes representing control and Sec61β depleted cells without EndoH treatment, the majority of Gurken protein runs as a single band along with minor lower molecular weight bands. (Fig. 5E, left half). Majority of the Gurken protein is also glycosylated since after EndoH treatment it runs at lower molecular weight (Fig. 5E, right half). It is important to note that the relative ratios of the glycosylated and unglycosylated forms of Gurken protein (1 g and 0 g respectively) does not change in the absence of Sec61β. Thus, the translocation of Gurken into the ER and post-translocational processes such as glycoslation are not affected by the reduced level of Sec61β. These experiments also show that cleavage of Gurken is unaffected by reduced amounts of Sec61β, suggesting that Rho is translocated and functional (Fig 5D).
Taking together the data from co-staining in the oocytes and experiments in HeLa cells it seems that Gurken translocation into the ER and its post-translational modifications remain unaffected by lack of Sec61β. Sec61β seems to affect a step beyond the process of ER translocation.
Gurken is transported till the Golgi complex in absence of Sec61β
Data obtained from immunofluorescence analysis in oocytes and in HeLa cells suggested that Sec61β was not involved in the transport of the Gurken protein into the ER, on the contrary it appeared that it is required at a later, post-ER step of secretion. The finding that lack of a component of the translocation channel could affect a step beyond the ER translocation was indeed surprising. In order to investigate this further and we wanted to analyse the sub-cellular localization of Gurken in greater detail. In both wild type and mutant oocytes the cytoplasmic pool of Gurken protein is present in the peri-nuclear region and is organised into punctate structures. These Gurken containing punctate structures are interspaced within the dispersed ER in both wild type and sec61β mutant oocytes. In order to establish the identity of these punctuate structures we used antibody against the Lava Lamp (Lva) protein, a cis-Golgi marker [19] together with the Gurken antibody for immunofluorescence analysis. We observed that the punctate structures containing the Gurken protein frequently also contained the Lva protein and this was visible in either the wild type (Fig. 6A–C) or the sec61βP1oocytes (Fig. 6D–F).
We also analysed the sub-cellular localization of the Gurken protein using a ubiquitously expressing an EYFP tagged Golgi marker [20]. In both wild type (Fig 7A–C) and sec61β mutant oocytes (Fig 7D–F) we observed that a significant number of the punctate Gurken structures in the cytoplasm co-localized with the Golgi marker.
This suggests that in the sec61β mutants Gurken protein is able to enter the secretory pathway and traffic till cis-Golgi, further transport to the plasma membrane however is impeded.
Sec5 retains plasma membrane localization in sec61βP1germline clones
Based on the data obtained so far it seemed that Sec61β is involved in a post-translocation step during trafficking of a subset of proteins to the plasma membrane. We therefore wanted to analyse the possible post-translocational steps in Gurken transport that could be affected by lack of Sec61β. Recent data suggests that Sec61β interacts with the components of the exocyst machinery in S. cerevisiae and in mammalian cells [21, 22]. The exocyst complex is a multi-protein vesicle-tethering complex localized at the plasma membrane that plays a role in polarised protein transport [23]. Directional transport of Gurken to the plasma membrane requires the exocyst complex. Germline clones of a hypomorphic allele of a subunit of the exocyst complex, sec5E13, shows lack of Gurken at the plasma membrane and cytoplasmic accumulation of Gurken [24]. Based on the interaction data and defects in Gurken localization in germline clones of sec5E13 and sec61βP1, we considered the possibility that failure of Gurken transport from the cytoplasmic location to the plasma membrane in Sec61β mutant egg chambers is due to lack of a functional exocyst.
In order to investigate this possibility we examined the localization of Sec5 protein in germline clones of sec61βP1. We used an antibody against Sec5 to stain the egg chambers during stage 10 of oogenesis. In wild type oocytes Sec5 is localized to the plasma membrane (Fig. 8A–C). In the oocytes from sec61βP1germline clones (Fig. 8D–E), the localization of Sec5 appears identical to the wild type oocyte. Thus, in absence of Sec61β, Sec5 retains its plasma membrane localization.