Canonical and potential non-canonical retention signals in pFGE 82

Based on the prototype KDEL, the various C-terminal tetrapeptide sequences can be categorized into KDEL-like canonical and sequences that differ from the KDEL motif (Fig. 3.5). 28 sequences in-group one contain a sequence of the K/R/H-X-D/E-L type (basic-X-acidic-leucine) at the C-terminus. In fact, the prototype KDEL itself was found in planarian pFGE (Schmidtea mediterranea) and KEDL in the murine orthologs (Mus musculusand Rattus rattus). However, human pFGE and pFGE from fifteen other species carried a PGEL sequence at the C-terminus, i.e. without a basic residue at the first or second position of the tetrapeptide but with the conservation of the terminal glutamate and leucine. Apart from the PGEL, other variants, mostly SGEL, were found in ten other species.

In earlier studies on the highly conserved sequence KDEL present at the C-terminus of the ER-retained protein, showed that this motif is necessary for re-tention and sufficient to confer rere-tention to a secretory protein [Munro and Pelham, 1987; Zagouras and Rose, 1989]. However, by amino acid substitution of KDEL sequence a number of closely related sequences were shown to confer retention to secretory proteins (Table 4.1) [Andres et al., 1990, 1991; Denecke et al., 1992].

Other amino acid substitutions can cause a complete loss of its function as a re-tention signal, demonstrating the high specificity of the rere-tention machinery (Table 4.1). However, naturally occurring variants were found in ER resident proteins for example, HNEL in LDL-receptor associated protein (RAP) [Bu et al.,1995], KEEL in

4.1. Is pFGE retention mediated by a new KDEL-like retention signal? 83 ERp72 [Mazzarella et al.,1990], HDEF in calumenin [Yabe et al.,1997] (Table 4.1).

In an overview (Table 4.1), the first position of the tetrapeptide can undergo

KDQL

Table 4.1: Variants of KDEL-like retention signals studied to mediate retention (for references, see text).

conservative changes to other basic residues like His/Arg, or to an acidic residue like Asp (DKEL, provided there is a basic residue in the second position). Further, we could rarely encounter an uncharged residue like Gln in the first position of the tetrapeptide. However, Ser in the first position of the tetrapeptide was found to abolish ER retention in plants [Denecke et al., 1992]. The second position of the tetrapeptide is relatively more relaxed hence it is represented as ’X’ in the canonical representation of basic-X-acidic-leucine motif of the ER retention signals (where X can be any residue). The third residue of the tetrapeptide is always a Glu or a Asp;

the fourth residue in most cases a leu, or rarely a Phe [Yabe et al., 1997]. Ile in position four does not impair retention in a substitution study [Andres et al.,1991]

but has not been found so far in ER proteins.

Therefore, the absence of a basic or a acidic residue at the first or the second position of PGEL and SGEL makes them a unique ER retention signal which does not fit to the canonical KDEL motif.

4.1.2 PGEL and SGEL tetrapeptide mediate ER retention

Deleting the PGEL tetrapeptide from the C-terminus of human pFGE or covering it by a His₆-tag led to a clear reduction in intracellular retention of pFGE. Our observation has been substantiated by two different experiments. First, the analysis of the kinetics of metabolically labeled pFGE showed clearly a reduction of pFGE retention when the C-terminal tetrapeptide PGEL has been removed. Moreover,

84 Chapter4. Discussion the rapid secretion of truncated pFGE during the pulse time suggested the loss of its retention determinant. The residual amount of truncated pFGE that is retained in the cell becomes secreted with time as evident in chase experiments.

Similarly, on truncation of its C-terminal retention signal KDEL, calreticulin was found to be secreted gradually [Sonnichsen et al.,1994]. Second, we could confer intracellular retention to lysozyme, a natural secretory protein by addition of PGEL to its C-terminus. Both experiments led us to draw the conclusion that PGEL is an autonomous retention signal similar to KDEL.

Further, our search in the protein database for any other proteins with PGEL at the C-terminus (Table 4.2) yielded four bacterial proteins and one hypothetical protein from Aspergillus. For none of these proteins, orthologs in metazoa could be found. Therefore, from this database search we can assume that C-terminal PGEL is found only in the ER-resident pFGE where it functions as an ER-retention signal.

Another potential, non-canonical tetrapeptide, SGEL, studied as a substitute for

Predicted protein

Table 4.2: Non-pFGE proteins with C-terminal PGEL identified in Swiss-Prot and TrEMBL.

PGEL in human pFGE, could also mediate ER retention to pFGE. Our search in the protein database for non-pFGE proteins with C-terminal SGEL yielded four proteins (as listed in Table 4.3). SGEL is found in cyclophilin proteins, which are known to be localized in the ER, Golgi and cell surface, functioning as a peptidyl-prolyl cis-trans isomerase and as chaperones [Price et al., 1999]. A putative peptidyl-prolyl cis-trans isomerase from amoeba has also been found. Another protein with C-terminal SGEL was found in Zebrafish. This homolog of Roundabout is a putative cell surface protein involved in axonal navigation, for which ER localization is more unlikely. As a sum, our data strongly suggests that C-terminal SGEL can function as a retention signal in higher and also lower eukaryotes.

From these studies we can add PGEL and SGEL as new ER retention signals

4.1. Is pFGE retention mediated by a new KDEL-like retention signal? 85

Table 4.3: Non-pFGE proteins identified by searching for C-terminal SGEL in Swiss-Prot and TrEMBL.

to the list of proved retention signals (Table 4.1). Based on this list we can now formulate the following new KDEL-like motif [KRHDQPS]-[DENKG]-[ED]-[LIF].

This motif includes all amino acids which have been proved by substitution or truncation experiments to be compatible with ER retention at their position in the sequence. It has to be noted that occurrence of these amino acids sometimes depend on the presence of other amino acids in the tetrapeptide. Further, some variants may function as retention signals in animals but not in plants and vice-versa (as shown for KDEI in Table 4.1).

4.1.3 Likely retrieval mechanism mediated by the PGEL signal