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Sorting of Mannose 6-Phosphate Receptors Mediated by the GGAs
Rosa Puertollano, Rubén C. Aguilar, Inna Gorshkova, Robert J. Crouch, and Juan S. Bonifacino

Supplementary Material

Additional details of recombinant constructs used in this study
The constructs GAL4ad-μ2 (1), GAL4bd-TGN38 (2), GAL4bd-LAMP-2 (3) and GAL4bd-ARF1 (Q71L) (4) have been described previously. The different GAL4ad-VHS constructs were prepared by ligating PCR cDNA fragments
encoding the VHS domains of HRS, STAM1, STAM2 (XmaI/Bam HI), TOM1L1 (XmaI/XhoI), TOM1, GGA1, GGA2, GGA3 (EcoRI/SalI) into the corresponding sites of the pGAD424(LEU2) vector (Clontech, Palo Alto, CA) (see ref. 4 for accession numbers for these proteins). The cDNAs corresponding to the cytoplasmic tails of mouse tyrosinase, TRP1, TRP2 (BamH1/Pst1), human transferrin receptor (EcoRI/PstI), β-Amyloid precursor (EcoRI/XhoI), LDL receptor, CI-M6PR (EcoRI/SalI), CD-M6PR (EcoRI/SalI) and Limp II (EcoRI/BamHI) proteins, were obtained by RT-PCR amplification with specific primers and then subcloned into the multiple cloning site of the pGBT9(TRP1) vector (Clontech). Constructs encoding the cytosolic tails of the CI-M6PR (residues 2378 to 2490) and CD-M6PR (residues 237-277) and these tails with deletions of the acidic-cluster-dileucine signals (residues 2378-2481 and 237-267 for CI-M6PR and CD-M6PR, respectively) were obtained by PCR-amplification and cloning into the EcoRI-SalI sites of pGEX-5X-1 (Pharmacia Biotech, Piscataway, NJ). The VHS domain of GGA1 (residues 1-147) was cloned in pET-28a(+) (Novagen, Madison, WI) via Eco R1 and Sal1 for production of a His6-tagged protein. In addition, the GGA1 full-length protein and the VHS-GAT region of GGA1 were cloned into the SalI and BamHI sites of both the pEYFP-C1 and pEGFP-C1 vectors (Clontech). The nucleotide sequences of all the recombinant constructs were confirmed by dideoxy sequencing.

Time-lapse imaging in living cells

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  • Movie 1
    Imaging of YFP-GGA1 in live MDCK cells. Time-lapse fluorescence video microscopy was performed using an Olympus 1X70 (Olympus, Hamburg, Germany) inverted microscope equipped with a polychrome II monochromator (TILL Photonics, Martinried, Germany). Data were processed to QuickTime format with Adobe Premiere 5.0 . Living cells expressing YFP-GGA1 were time-lapse recorded (0.5 s per frame) for 2 min. Note that vesicular structures containing YFP-GGA1 are continuously emerging from the Golgi complex and move toward the periphery with speeds of 1-4 μm/sec (arrows).

Supplementary References

  1. H. Ohno et al., Science269, 1872-1875 (1995).
  2. H. Ohno, M. C. Fournier, G. Poy, J. S. Bonifacino, J. Biol. Chem.271, 29009-29015 (1996).
  3. R. C. Aguilar et al., J. Biol. Chem.276, 13145-13152 (2001).
  4. E. C. Dell'Angelica et al., J. Cell Biol.149, 81-94 (2000).