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Address correspondence to Francis Barr, Dept. of Cell Biology, Max-Planck-Institute of Biochemistry, Am Klopferspitz 18a, 82152 Martinsried, Germany. Tel.: 49-89-8578-3135. Fax: 49-89-8578-3102. E-mail: barr{at}biochem.mpg.de
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Abstract |
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Key Words: GRASPs; p24 proteins; Golgi matrix; protein transport; cargo receptors
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Introduction |
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The most studied components of the Golgi matrix are p115, the GRASP65GM130 complex, and an integral membrane protein, giantin (Linstedt and Hauri, 1993; Nakamura et al., 1995; Sapperstein et al., 1995; Barr et al., 1997; Shorter and Warren, 1999). GRASP65 was identified in a screen for factors involved in the stacking of cisternae, and later shown to be a specific binding partner of GM130 required to target it to the Golgi (Barr et al., 1998). GM130 in turn is a receptor for p115, required for tethering vesicles to their target membrane (Barroso et al., 1995; Nakamura et al., 1997). More recently, the formation of Golgi stacks from cisternae in vitro was found to require p115, discrete from its membrane fusion function, and giantin (Shorter and Warren, 1999). GM130 and p115 also make interactions with giantin during vesicle docking, and potentially during cisternal stacking (Shorter and Warren, 1999; Dirac-Svejstrup et al., 2000; Lesa et al., 2000). The network of interactions between these proteins may be regulated by the rab GTPases, as both p115 and GM130 have been shown to interact with the active or GTP form of rab1 (Allan et al., 2000; Moyer et al., 2001; Weide et al., 2001). Recruitment of p115 to vesicles destined to fuse with the cis-Golgi is mediated via the interaction with rab1 (Allan et al., 2000), whereas the consequences of rab1 binding to GM130 are unknown. A second GRASP complex, containing GRASP55 and the coiled-coil protein golgin-45, exists in Golgi membranes (Shorter et al., 1999; Short et al., 2001). Like the GRASP65GM130 complex, this complex also binds a rab GTPase and is required for the maintenance of normal Golgi structure and protein transport (Short et al., 2001).
In addition to the Golgi matrix proteins discussed above, some membrane proteins are of potential importance to Golgi structure. One such group is the medial-Golgi enzymes, found to specifically bind to the Golgi matrix although the mechanism remains uncharacterized (Slusarewicz et al., 1994). Another is the p24 family of cargo receptors, identified as major transmembrane components of vesicles recycling between the ER and Golgi complex, and as potential structural Golgi proteins in mammalian cells (Wada et al., 1991; Stamnes et al., 1995; Rojo et al., 1997). All p24 proteins share a common structure, with a short cytoplasmic domain containing binding signals for the COP-I and COP-II vesicle coat complexes, plus a lumenal domain with potential secretory cargo binding capabilities (Fiedler et al., 1996; Sohn et al., 1996; Dominguez et al., 1998; Muniz et al., 2000). At least one p24 protein, TMP21, is an essential gene in mammals, and a heterozygous deletion shows reduced levels of the protein and a partially disrupted Golgi apparatus (Denzel et al., 2000). However, in yeast, all p24 family members can be deleted with little or no effect on protein transport or secretory pathway morphology (Springer et al., 2000).
The drug brefeldin A disrupts the Golgi apparatus and causes Golgi enzymes, but not putative structural components such as the GRASPs and GM130, to be relocated to the ER (Nakamura et al., 1995; Seeman et al., 2000). Under conditions in which the Golgi is first disrupted with brefeldin A and then allowed to recover while protein transport from the ER is blocked, a Golgi-like structure forms that lacks Golgi enzymes but contains Golgi matrix proteins (Seeman et al., 2000). However, under the electron microscope, these structures lack some key features of normal Golgi membranes, such as well-defined stacked cisternae. Therefore, the Golgi matrix must interact with additional factors, either integral membrane proteins or specific lipids to organize Golgi membranes. Therefore, we decided to investigate whether GRASPs could provide a link between Golgi matrix components and transported integral membrane proteins.
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Results and discussion |
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The transmembrane precursor of TGF- also binds to GRASP55 in the Golgi, an interaction necessary for its correct localization and transport (Kuo et al., 2000). We show that GRASP65 can also bind to the TGF-
cytoplasmic domain, raising the possibility that GRASPs could bind to a variety of transmembrane cargo proteins via their cytoplasmic domains. Therefore, interaction with Golgi matrix proteins may be a general sequestration mechanism to regulate the transport and retention of integral membrane proteins within the Golgi apparatus.
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Materials and methods |
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Purification and analysis of GRASP55 complexes
GRASP55 complexes were purified as described previously (Short et al., 2001). Aliquots of total extract and bound protein complexes were analyzed by SDS-PAGE on 10% minigels under nonreducing conditions. Proteins were extracted from Coomassie bluestained gel slices and digested with sequencing-grade trypsin (Promega) for analysis of the peptide fragments by MALDI-TOF (Reflex III; Bruker) and probability-based database searching (Perkins et al., 1999).
Yeast two-hybrid and protein binding assays
Standard molecular biology techniques were used for the construction of all constructs, and all constructs were confirmed by DNA sequencing (Medigenomix). Double-stranded oligonucleotides corresponding to the cytoplasmic domains of the p24 proteins, TGF-, and the COOH terminus of GM130 were inserted into the EcoRI and BamHI sites of pGBT9. Coiled coilcontaining constructs were made by inserting a PCR-amplified fragment corresponding to amino acids 755974 of GM130 into the EcoRI site of pGBT9. The full-length rat GRASP55 and GRASP65 constructs in pACT2 were described previously (Shorter et al., 1999). Yeast two-hybrid assays were performed as described previously (Short et al., 2001). Measurements of lacZ reporter gene expression were performed according to the CLONTECH Yeast Protocol handbook, and the results are expressed in Miller units (Miller, 1992). Bacterial hexahistidinetagged GST fusion protein constructs for p24, TGF-
, and GM130 were constructed by transferring the EcoRI and HindIII fragment containing the various tail sequences from pGBT9 to pGAT2. Hemaglutinin epitope GRASP55 and untagged GRASP65 were cloned into pQE32 for bacterial expression. Proteins were expressed in BL21(DE3) for pGAT2 constructs at 37°C for 3 h, or JM109 for pQE32 constructs at 23°C for 18 h. Fusion proteins were purified on nitrilotriacetic acidnickel agarose according to the manufacturer's instructions (QIAGEN), dialyzed against PBS, and then stored at -80°C. For each binding assay, 5 µg of GST fusion protein was bound to 10 µl glutathione-Sepharose (Amersham-Pharmacia Biotech). These beads were incubated in the presence of 10 ng/µl GRASP55 or GRASP65 in a total volume of 200 µl PNT (25 mM sodium phosphate, pH 7.2, 100 mM NaCl, 0.1% TX-100) for 1 h at 4°C on a rotating wheel. Beads were washed three times with 400 µl PNT, and then bound proteins eluted with sample buffer for analysis by SDS-PAGE and Western blotting. Bound GRASP proteins were detected using a monoclonal antibody to the hemaglutinin epitope or the 7E10 monoclonal antibody to GRASP65, and GST fusion proteins with a polyclonal antibody to GST.
Microscopy and protein transport assays
GFP-tagged p24a expression constructs were made by amplification of the p24a lumenal and transmembrane domains using primers that replaced the cytoplasmic domain with the required mutant sequence. These fragments were then cloned back into the GFP-p24 expression vector (Blum et al., 2000). HeLa cells plated on glass coverslips were transfected with a plasmid encoding GFP-tagged p24a, or mutants thereof, for 18 h at 37°C. The cells were then fixed with 4% paraformaldehyde in PBS. Cell surface p24a was detected with a sheep antibody to GFP attached to the lumenal domain and a donkey antisheep secondary coupled to CY3 (Jackson ImmunoResearch Laboratories), and total p24a by GFP fluorescence. Images were collected using a Zeiss Axioskop-2 with 63x Plan Apochromat objective equipped with a 1300 by 1030 pixel cooled-CCD camera (Princeton Instruments), and Metaview software (Universal Imaging Corp.). The ratio of surface to total measured fluorescence was used to normalize the amount of p24a at the cell surface.
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Footnotes |
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Acknowledgments |
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Research in the laboratory of F.A. Barr is supported by the Max-Planck-Gesellschaft (München/Germany).
Submitted: 20 August 2001
Revised: 15 October 2001
Accepted: 23 October 2001
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References |
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