3.5 Significance of FGE N-terminus for its retention
3.5.4 Co-elution of Gal6S with FGE-His 6 or ∆34-68 FGE-His 6
FGE is known to form complexes with its substrate galactose-6-sulfatase, that persist on size exclusion chromatography [Mariappan, 2005]. It was surprising to see a significant reduction of the in vivo but not in vitro activity of ∆34-68 FGE-His6, leaving with the question whether FGE-His6 but not ∆34-68 FGE-His6 is able to
78 Chapter3. Results
Gal6S specific activity vs FGE or Δ34-68 FGE
0
0 500 1000 1500 2000
FGE or Δ34-68 FGE (ng/mg total cell protein) FGE + Gal 6S Δ34-68 FGE + Gal6S Gal6S specific activity (µmol/µg Gal6S)
Figure 3.32: Specific activity of Gal6S depends on the amount of FGE.Specific activity of Gla6S was measured in different clones coexpressing FGE-His6 or∆34-68 FGE-His6 (Fig. 3.31).
The amount of Gal6S, FGE-His6 and ∆34-68 FGE-His6 in these clones were quantified by Western blot (Fig. 3.30). The specific activity was plotted against the amount of FGE-His6 or
∆34-68 FGE-His6 in the cells.
form a complex with the Gal6S. To identify complexes we have done a size exclusion chromatography with the cell extracts prepared from the cells stably expressing either Gal6S alone or ∆34-68 FGE-His6 and Gal6S (clone A3) or FGE-His6 and Gal6S. Monomers of Gal6S are eluted in fractions 16 to 21 (Fig. 3.33upper panel), and of FGE-His6 or ∆34-68 FGE-His6 in fractions 18 to 21 (not shown). In cells coexpressing Gal6S with FGE-His6 or with∆34-68 FGE-His6 complexes of FGE-His6 and Gal6S or ∆34-68 FGE-His6 and Gal6S were detected in fractions 15 and 16 (Fig. 3.33 middle and lower panel respectively). This indicates that the complex
Gal6S and
Figure 3.33: Elution profile of Gal6S and coelution profile of Gal6S and∆34-68 FGE-His6 or FGE-His6. Cell extracts from cells stably expressing Gal6S (upper panel) or FGE-His6 and Gal6S (middle panel) or∆34-68 FGE-His6and Gal6S (lower panel) were loaded on Superdex-200 as described in materials and methods. The fractions were analysed by immunoboltting with a mix of Gal6S monoclonal antibody and FGE polyclonal antibodies.
formation with Gal6S does not involve the N-terminus of FGE.
3.5. Significance of FGE N-terminus for its retention 79
19 FT CoIP
Gal6S and ∆34-69 FGE-His6 (A3) Gal6S and FGE-His6(B3)
15 FT CoIP
Fraction No.
FGE-His6
Gal6S
Gal6S
∆34-69 FGE-His6
Figure 3.34: Co-immuno precipitation of Gal6S with FGE. Specificity of the Gal6S and FGE or Gal6S and∆34-68 FGE complexes was tested by co-immuno precipitation of Gal6S from the co-eluted fractions (14 to 16 and 19 to 21) using with FGE polyclonal antibodies.
To verify that co-elution of ∆34-68 FGE-His6 or FGE-His6 with Gal6S is due to their complex formation, we have done the co-immunoprecipitation (Co-IP) of the fractions 14, 15, 16 and fractions 19, 20, 21 using FGE specific polyclonal antibodies. The results showed that both FGE-His6 and ∆34-68 FGE-His6 were pulled down together with Gal6S only in the fraction 15 (Fig. 3.34). Therefore, the co-elution of ∆34-68 FGE-His6 and Gal6S or FGE-His6 and Gal6S in the early fractions is due to complex formation by specific interactions.
In summary, we can conclude that the amino-terminal truncation impairs the retention and in vivo activity of FGE, without influencing its in vitro activity and binding to galactose-6-Sulfatase.
Chapter 4 Discussion
The identity of the ER is maintained by a concerted and balanced action of three mechanisms: correct sorting of the proteins to be exported, retention of the resident proteins and retrieval/recycling of the escapees. Among these the best understood is the retrieval/recycling of the soluble ER proteins bearing H/KDEL-type reten-tion signals [Munro and Pelham, 1987; Pelham, 1988, 1990] and the retrograde transport of membrane proteins bearing cytosolic KKXX or RR motifs or a luminal HDEL [Teasdale and Jackson, 1996]. But exceptions to the general rule do exist.
Some membrane proteins bearing the ER retention signals are not localized in the ER, e.g., ERGIC 53 [Schindler et al., 1993]. Moreover the C-terminal domain of calreticulin and BiP were shown to participate in their retention possibly by a (calcium dependent) interaction with the reticular matrix [Munro and Pelham, 1987;Zagouras and Rose,1989]. A good evidence for the exception to the general rule comes from the continuous discovery of soluble ER-resident proteins, such as human FGE and pFGE, without any canonical retention signals.
In the present study we determined the ER retention mechanism of pFGE, the paralog of formylglycine-generating enzyme (FGE) and explored the possible ER retention mechanisms of FGE.
4.1 Is pFGE retention mediated by a new KDEL-like retention signal?
When we studied the effect of expression levels on the retention of untagged pFGE, we observed that with increasing levels of expression there is a relative decrease in the retention of pFGE, and that overexpression resulted in its anterograde transport along the secretory route. This led us to infer that pFGE is likely to be retained
81
82 Chapter4. Discussion in the ER by a saturable mechanism, in accordance with a previous report on the saturation of the ER retention machinery by overexpression of ER-resident proteins such as calreticulin or PDI. [Crofts et al.,2000;Monnat et al.,1999].
Previous studies in our group had reported that endogenous pFGE was retained almost exclusively in the ER, whereas recombinant pFGE with a C-terminal tag was localized to the ER, to the Golgi and even secreted. In this study we have observed that the saturable retention mechanism of pFGE is mediated by its determinant at the C-terminus. When we expressed wt-pFGE and pFGE-His6 we have found that the C-terminal tag is interfering with efficient retention of pFGE which can be substantiated by the previous observation on the massive secretion of pFGE-His6 [Mariappan et al.,2005]. Accordingly, we inspected the protein sequences of pFGE from all the available species and found one group of species containing a KDEL-like C-terminal sequence, a second group, however, including human pFGE with C-terminal tetrapeptides different from the KDEL motif.