Indeed,in vitropFGE shows no FGly generating activity on all the peptides derived from 16 sulfatases under conditions appropriate for the FGly generating activity of FGE [Mariappan et al.,2005].
Interestingly, in vivo overexpression of pFGE interferes with FGly formation in sulfatases [Mariappan et al., 2005]. This effect may be due to binding of sulfatases to pFGE, thereby sequestering them from FGE. However, the effects of pFGE on FGE are also conceivable in an indirect manner, e.g. by dislocating FGE from the endoplasmic reticulum (ER) due to competition for a common retention mechanism, or directly by heterodimerization.
1.4 ER retention mechanisms
Endoplasmic reticulum constitutes a major compartment of the complex endomem-brane system that makes up the secretory pathway. It is the central port for the entry of proteins into the secretory pathway for their distribution to different target organelles via trans-Golgi network or their secretion. It provides an optimized environment favorable for proper protein folding and maturation. The co- or post-translational modifications of secretory, luminal and integral membrane proteins are facilitated by soluble and membrane-bound ER-resident proteins such as BiP, calreticulin, calnexin and PDI. They function not only as chaperones but also as the components of quality control machinery for the forward transport of the proteins, by serving as retention anchors for immature proteins. The transport-competent mature cargo protein usually corresponds to the compactly folded native conformation that has undergone correct co- or post-translational processing. Cargo proteins need to be sorted from the ER-resident proteins by gaining access to the COPII-coated vesicle-forming site of the ER, designated as ER exit site or transitional ER.
Packing of the cargo proteins into the budding vesicles from the ER could be by bulk flow or by a selective process. Some soluble and membrane cargo proteins are actively recruited into the vesicles where they become concentrated, this enrichment is achieved by interaction of the cytoplasmic coat with distinct sorting signals on the cytoplasmic domain of the membrane protein. However, soluble cargo protein and GPI-anchored membrane proteins that do not present any cytoplasmic signal interact with specific trans-membrane receptors that serve to link these luminal substrates to the cytoplasmic coat e.g. lectin chaperones such as calnexin and calreticulin and ERGIC53. Moreover, at the site of budding vesicles
14 Chapter1. Introduction ER-resident soluble proteins without exit signals can also get packaged. Similarly, high concentrations of secretory proteins may facilitate export without the help of sorting receptors.
Sar1, a small GTP-binding protein of the COPII coatomer complex assembly at the budding vesicle, enables delivering the cargo proteins from the ER to Golgi by anterograde transport via the putative intermediate compartment formed by the vesicular tubular clusters (VTC) also known as ER-Golgi intermediate compartment (ERGIC).
1.4.1 ER retention by retrieval
ER-resident proteins in the VTC or Golgi that escaped ER retention are retrieved back to the ER in the retrograde transport from Golgi, mediated by COPI coatomer complex and ADP ribosylation factor (ARF), a small GTP-binding protein based on the ER retrieval signals. Resident ER membrane proteins bear a specific retrieval signal, which act as sorting signal. The well-characterized sorting signal is the di-lysine motif found at the C-terminus of type I transmembrane proteins (KKXX or KXKXX, where X is any amino acid). The di-lysine motif shows direct interaction with specific subunits of the COPI coatomer complex that mediates retrieval to ER from Golgi [Letourneur et al., 1994]. Similarly, di-arginine motifs (RR) found at the N-terminus of type II transmembrane proteins are also retrieved by COPI, recognized by different binding pockets of the coat complex. Whereas soluble ER-resident proteins bear a specific retrieval signal (KDEL in mammals, HDEL in yeast) that mediate interaction with the KDEL receptor (ERD2 in yeast and the homologous human receptors designated as KDELR1 and KDELR2 also known as hERD2.1 and hERD2.2 respectively) [Lewis and Pelham, 1990, 1992; Semenza et al., 1990].
KDELR3 is the third member of the family to be identified, and it encodes a protein highly homologous to KDELR1 and KDELR2 proteins. Two transcript variants of KDELR3 arise by alternative splicing, and encode different isoforms of KDELR3 re-ceptor. The KDEL receptor itself presents a cytoplasmic di-lysine retrieval motif that contributes to interaction with the COPI coat in conjugation with a phosphoserine residue that is also important for ER-Golgi retrieval [Cabrera et al.,2003]. Although the precise mechanism by which KDEL receptor binds its ligands is not known, a number of point mutations on the luminal surface of the receptor inhibits binding to KDEL peptides, implying that these residues contribute to a ligand binding pocket [Scheel and Pelham, 1998]. Furthermore, ligand binding to the KDEL receptor is thought to leads to conformational changes that trigger uptake of the assembly complex into ARF and COPI vesicles [Lewis and Pelham, 1992]. This regulated
1.4. ER retention mechanisms 15 transport may be driven by the ligand-induced oligomerization of the receptor, which in turn stimulates interaction with COPI coat complex [Majoul et al.,2001].
Biochemical characterization of the receptor showed that it specifically binds the ligand in pH-dependent manner, suggesting that subtle differences in the luminal environment will allow release of KDEL-containing proteins upon fusion with the ER [Wilson et al.,1993].
1.4.2 Direct ER retention
Some of the endogenous proteins are found to lack detectable post-ER modifications like calreticulin, a luminal ER protein with C-terminal KDEL, and UDP-glucuronyl-transferase, an ER type I membrane protein with di-lysine motif. ER retention of calreticulin was suggested to occur by a KDEL-based retrieval system and by a calcium dependent direct retention [Sonnichsen et al., 1994]. Moreover, there is evidence that the resident ER proteins form a dynamic network stabilized by weak interactions modulated by the high lumenal calcium ion concentration [Kreibich et al.,1978]. Such a matrix may contribute to the retention of incompletely folded proteins [Tatu and Helenius,1997].
1.4.3 Thiol-mediated ER retention
A special mechanism of ER retention involves exposed free cysteines. This was first described for the retention of unassembled immunoglobulin chains [Sitia et al., 1990] by forming intermolecular disulfide-bonding with ER-resident thiol oxidore-ductases such as PDI and ERp72 [Reddy and Corley, 1998]. Thiol mediated retention has also been shown for unassembled subunits of acetylcholinesterase [Kerem et al., 1993] and Ero1 (an oxidoreductase that lacks known ER retention motifs). Recently, it was found that ERp44 mediates ER localization of Ero1 or IgM subunits by formation of reversible mixed disulfides.
1.4.4 Retention by aggregation
Newly synthesized proteins may also be retained in the ER by mutual interactions with each other, resulting in the formation of large aggregates which are transport-incompetent. Many of the aggregates are cross-linked by non-native interchain disulfide bonds. Thyroglobulin, major histocompatibility complex (MHC) class II, and procollagen form transient aggregates before acquiring their native structures.
16 Chapter1. Introduction
1.4.5 Other possible retention mechanisms
Some of the resident ER proteins are found to lack canonical retention signals and to be retained by formation of hetero-oligomers with proteins containing H/KDEL-containing proteins. Mouse liver-Glucuronidase is retained within the ER via complex formation with esterase-22 (egasyn), which in turn has a carboxyl-terminal HTEL ER-retention sequence [Zhen et al.,1995].
In summary, exceptions to the general ER retention mechanisms do exist paving path for the identification of new retention or sorting signals that are essential for the correct localization of the proteins to places where their biological activity is required.