Department of Medical Microbiology and Immunology, University of Alberta, Edmonton, Alberta T6G 2S2, Canada
Received on August 19, 1999; revised on November 1, 1999; accepted on December 8, 1999.
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Abstract |
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Key words: ER quality control/glucosidase II/glycoprotein processing/N-glycosylation
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Introduction |
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Interestingly, many of these insights into the central function of GII in glycoprotein maturation were acquired before the true molecular identity of the protein was known. In a recent attempt to reconcile this, sequence information was obtained on GII purified from rat liver as a heterodimer of two noncovalently linked but strongly associated subunits (GII and GIIß) (Trombetta et al., 1996
). In addition, we recently isolated the mouse cDNAs encoding these two proteins following their copurification with the transmembrane protein-tyrosine phosphatase CD45 from T-cell lysates (Arendt and Ostergaard, 1997
). Homology searches revealed that mouse GII
and a recently cloned pig homologue (Flura et al., 1997
) share, respectively, 90% and 92% protein sequence identity to a human cDNA clone in the GenBank database (Nagase et al., 1995
). GII
lacks a transmembrane segment and a known retention/retrieval motif, however the mouse ß-subunit coding sequence has a C-terminal His-Asp-Glu-Leu (HDEL) sequence (Arendt and Ostergaard, 1997
) that is conserved in its highly homologous human counterpart (Sakai et al., 1989
). Since this motif has been shown to be sufficient for recognition by the KDEL receptor (Ozawa and Muramatsu, 1993
; Wilson et al., 1993
), it has been postulated that GIIß functions to couple the
-subunit to this ER-retrieval mechanism (Trombetta et al., 1996
). Interestingly, GIIß also possesses two putative EF hand domains and a region of negatively charged repeats, features thought to confer Ca2+ binding properties to other ER proteins (Booth and Koch, 1989
; Sonnichsen et al., 1994
; Weis et al., 1994
). As there is evidence for the involvement of a calcium matrix in the retention of resident ER proteins (Booth and Koch, 1989
; Sonnichsen et al., 1994
; Weis et al., 1994
), it is possible that GIIß invokes multiple mechanisms to ensure localization of GII
to the endoplasmic reticulum.
In the present study, we have utilized a panel of GIIß fusion proteins to identify functional domains that mediate binding of this protein to the catalytic subunit. The results of our analysis indicate the presence of two distinct interaction domains, ID1 and ID2, in GIIß. We hypothesize that synergy in the binding activity of these two sites accounts for the highly stable intermolecular association of the - and ß-subunits of the GII heterodimer.
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Results |
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Discussion |
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We sought to gain insight into the function of GIIß by defining the regions of the molecule that interact with the catalytic subunit of GII. By generating a large panel of native recombinant GST-fusion proteins, we identified two distinct interaction domains, designated ID1 and ID2, that are capable of associating with GII. ID1 is an N-terminal stretch of 118 amino acids that terminates well upstream of the putative EF-hand domain of GIIß. Although ID1 spans the entire N-terminal Cys-rich element of GIIß, disulfide linkages do not mediate its interaction with the
-subunit, since the precipitation of GII
by GSTß(N-118), as visualized on Coomassie stained gels, is not significantly impaired by addition of 20 mM iodoacetamide to the lysis buffer (data not shown). This is consistent with a previous study that failed to detect covalent intermolecular linkages between the two subunits of GII (Trombetta et al., 1996
). In contrast to the N-terminal location of ID1, ID2, as defined by our mapping studies, is contained within residues 273400 of GIIß. The region encompassed by ID2 contains an acidic stretch flanked by proline-rich elements but excludes the C-terminal Cys-rich element. Although a segment of 7 variably expressed amino acids is located near a 127 amino-acid region of ID2 possessing a critical binding determinant (Figure 5), our binding studies failed to support a role for these 7 residues in modulating the association with the
-subunit. However, we cannot exclude the possibility that this segment influences the interaction of the two subunits in vivo in the context of full-length GIIß.
That our experiments implicate not one but two domains in GIIß in the interaction with GII is consistent with biochemical evidence that the
- and ß-subunits of GII are highly refractory to dissociation (Trombetta et al., 1996
). Analysis of purified GII by sucrose gradient fractionation, gel filtration, and nondenaturing polyacrylamide electrophoresis suggests that the two subunits exist as a 1:1 heterodimeric complex (Trombetta et al., 1996
). Together, the available data suggest a model whereby ID1 and ID2 on a single molecule of GIIß, while individually capable of associating with the
-subunit, synergize in their binding interaction with a single molecule of GII
. While it is unlikely that ID1 and/or ID2 encode homooligomerization domains per se, GII heterodimers may closely interact with one another and other ER proteins as part of a larger protein network (Tatu and Helenius, 1997
).
The results of this study derive from observations with multiple GST-fusion proteins encompassing ID1 and ID2 and two antisera directed at other portions of the molecule. Since GII is a soluble enzyme, it should be possible to derive crystals for structural analysis of molecular interactions occurring between the - and ß-subunits. It will be particularly interesting to determine whether ID1 and ID2 are juxtaposed in the native structure of GIIß to form a contiguous binding interface that might interact with a single domain of GII
. Interestingly, ID1 superimposes the Cys-rich element at the N-terminus of GIIß, while ID2 is located adjacent to the C-terminal Cys-rich element. Reducing versus nonreducing gel analysis of purified GII has shown that intramolecular disulfide bonds are present in the ß-subunit (Trombetta et al., 1996
). It will be important to determine whether such linkages serve to bring the N- and C-termini of GIIß into close apposition.
In summary, we have provided the first biochemical demonstration that GIIß possesses two independent binding domains capable of coupling the molecule to the -subunit of GII. At this point we can only speculate that the role of this tightly associated ß-chain is to maintain GII
in the endoplasmic reticulum. Another enticing possibility is that GIIß may also exert a regulatory role upon the physical and functional interplay between GII and other members of the quality control apparatus whose activities are so closely coordinated.
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Materials and methods |
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Generation of GST fusion proteins
Flanking primers were designed to introduce an upstream EcoRI restriction site and a downstream in-frame stop codon followed by an XhoI restriction site. Inserts were amplified using the high fidelity Advantage cDNA Polymerase Mix (Clontech) by polymerase chain reaction from GIIß cDNA clones obtained previously (Arendt and Ostergaard, 1997). For most reactions, the thermokinetic parameters were 1 cycle of 1 min at 94°C followed by 2530 cycles of 30 s at 94°C, 1 min at 54°C, and 50 s at 68°C. Polymerase chain reaction products were cloned into the GST expression vector pGEX-4T-3 (Pharmacia) followed by transformation of E.coli strain JM105. GST-fusion protein expression was induced in log-phase bacterial cultures by addition of 0.1 mM isopropyl-ß-D-thiogalactopyranoside (Boehringer Mannheim) for 2 h. Bacteria were lysed by sonication in PBS containing 1 mM Pefabloc (Boehringer Mannheim), and GST-fusion proteins were purified by batch elution from glutathione-agarose matrix (Sigma) according to the instructions supplied with the vector. Following extensive dialysis against PBS, protein concentrations were measured by BCA assay (Pierce). Recombinant proteins were adjusted to 0.5 mg/ml and stored at 70°C at for later use. Purity of fusion proteins was assessed by subjecting samples to SDSPAGE on 10% gels followed by staining with Coomassie brilliant blue dye (Bio-Rad). The DNA sequence fidelity of the following constructs was confirmed by automated sequencing: GSTß(N-118), GSTß(N-98), GSTß(20258), GSTß(273400), GSTß*(273400), and GSTß(273344).
Binding assays
Cells were lysed at a density of 5 x 107/ml in 0.5% Nonidet P-40/1 mM Pefabloc/phosphate-buffered saline, pH 7.2 and 10 µg of GST-fusion protein or 5 µl of antiserum was added to 1 ml of postnuclear lysate. Following a 30 min incubation on ice, fusion proteins were captured by addition of 50 µl of a 50% slurry of glutathione-agarose while antibodies were captured by addition of the same volume of Protein A beads (Boehringer Mannheim). Samples were placed on a rotator at 4°C for 90 min, after which beads were washed three times with 1 ml of lysis buffer. Beads were then incubated on a rotator for 15 h at room temperature with 125 µl of a reaction mix consisting of 5 mM p-nitrophenyl -D-glucopyranoside (Sigma) in phosphate-buffered saline, pH 7.2. As a positive control, 2.5 µl to 25 µl of the original cell lysates were incubated with the same reaction mix. Color change was quantitated by transferring 100 µl from each tube to a 96-well plate and measuring absorbance at 405 nm. Background absorbance, defined as the average OD405 value obtained when pull-downs were carried out in lysis buffer alone, was subtracted from all values obtained. In all experiments, assays were performed independently in triplicate and mean values were graphed with error bars signifying standard error of the mean.
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Acknowledgments |
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Abbreviations |
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Footnotes |
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References |
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