Supplemental Data

Sortilin Is Essential and Sufficient

for the Formation of Glut4 Storage Vesicles

in 3T3-L1 Adipocytes

Jun Shi and Konstantin V. Kandror

Supplemental Experimental Procedures

Plasmids in Retroviral Vectors

The pLNCX2 vector was obtained from BD Clontech (Palo Alto, CA) and the pBabe-puro retroviral vector was a kind gift from Dr. Stephen R. Farmer (Boston University School of Medicine). The pcDNA3.1/myc-His A vector was obtained from Invitrogen (Carlsbad, CA). A human Glut4 cDNA containing 7-myc epitopes in the first extracellular loop (a kind gift from Dr. Jonathan S. Bogan, Yale School of Medicine) was subcloned into the pcDNA3.1(+) vector as described earlier (Bogan et al., 2001). The human sortilin cDNA construct pcDNA3.1Zeo(-)-sortilin (NCBI accession number HSGP95SOR) was a kind gift from Dr. Peder Madsen and Dr. Claus Petersen (University of Aarhus, Denmark). For the preparation of retroviral vectors with Glut4, two oligos (both from MWG Biotech, High Point, NC), 5-TCGAGGGATCCGTTTAAACGC-3 and 5-GGCCGCGTTTAAACGGATCCC-3, were annealed and ligated to the XhoI and NotI cut pLNCX2 vector. The modified vector called mLNCX2 contained BamHI and PmeI sites between XhoI and NotI sites instead of the original HindIII and SfiI sites. In order to obtain mLNCX2-myc7-Glut4, the BamHI/NotI fragment from pcDNA3.1-myc7-Glut4 was cloned into mLNCX2 retroviral vector. In order to obtain pBabe-myc7-Glut4, a BamHI/SalI fragment from mLNCX2-myc7-Glut4 was cloned into pBabe-puro. For the preparation of retroviral vectors with sortilin, the 2.5 kb XhoI/ApaI fragment including the whole open reading frame of sortilin was amplified by PCR using primers 5-ACGACGTCACTCGAGATGGAGCGGCCCTGGGGAG-3 and 5-CTAGACCTGGGGCCCTTCCAAGAGGTCCTC-3. This fragment was ligated into the corresponding sites of the pcDNA3.1/myc-His A vector. The XhoI/PmeI fragment, which included sortilin open reading frame with myc-His tag at the C terminus, was inserted into the corresponding sites of the mLNCX2 vector, creating mLNCX2-sortilin-myc/His.

Plasmids in Lentiviral Vectors

The pLenti6/V5-D-TOPO cloning kit was purchased from Invitrogen (Carlsbad, CA). Two oligonucleotides (5-CACCGAATTCGTTTAAACTGTCGAC-3 and 5- GTCGACAGTTTAAACGAATTCGGTG-3) were annealed and ligated to the pLenti6/V5-D-TOPO vector. This modified vector is called pLenti vector. Two more oligonucleotides (5-GATCCGTTAACGAATTCTCGCTCGAGCCAGTGTGGTGGGTTTAAACTCTGCAGACTAGTGTCGACAGGGGCCCGC-3 and 5-GGGCCCCTGTCGACACTAGTCTGCAGAGTTTAAACCCACCACACTGGCTCGAGCGAGAATTCGTTAACG-3) were annealed and ligated to the BamHI and SacII cutted pLenti vector. This vector is called pLenti-m1. Full-length sortilin was released from mLNCX2-sortilin-myc/His by XhoI and PmeI and ligated to the corresponding sites of the pLenti-m1 vector, creating pLenti-m1-sortilin-myc/His. The fragment (nucleotides 1816-2349) of human sortilin was amplified with two primers: 5-GATTTTAAAGATATCCTTGAAAGGAA-3 and 5-TTGTGAAGAAATATGTCTGTCATCATCACCATCACCATACTAGT-3 (encoding the His tag), digested with EcoRV and SpeI and ligated to the corresponding sites of pLenti-m1-sortilin-myc/His. This vector (pLenti-sortilin-His/V5) encodes sortilin with the His/V5 tag instead of the original C terminus (starting from the sixth amino acid after the transmembrane domain).

EGFP was amplified from the pEGFP-C2 template using two primers 5-ACGACGTCAGGATCCGTCGCCACCATGGTGAGCAA-3 and 5-TCGAGTCTGGAATTCCTTGTACAGCTCGTCCATGCC-3. This fragment was digested by BamHI and SalI and ligated into the corresponding sites of pLenti-m1. The resulting vector is called pLenti-m1-EGFP-C2.

EGFP fragment was amplified from the pEGFP-N3 template using two primers 5-ACGACGACAGTCGACATCGCCACCATGGTGAGCAAG-3 and 5-CTGAGTCTGCCGCGGTTACTTGTACAGCTCGTCCATG-3. This fragment was digested sequentially by SalI and SacII and ligated into the corresponding sites of pLenti-m1. The resulting vector is called pLenti-m1-EGFP-N3.

The C terminus of human sortilin (nucleotides 2243-2496) was amplified using two primers 5-CAGCAGTCACTCGAGAAACAGAATTCCAAGTCAAATTCT-3 and 5-TCGAGTCTGACTAGTCTATTCCAAGAGGTCCTCATCT-3. This fragment was cut with XhoI and SpeI and ligated into the corresponding sites of pLenti-m1-EGFP-C2 vector, creating pLenti-m1-EGFP-mSorC.

Two oligoes, 5-GATCCGCCGCCACCATGTACCCATACGATGTTCCAGATTACGCTCTTGGGGGTTCTCATCATCATCATCATCATGGTG-3 and 5-AATTCACCATGATGATGATGATGATGAGAACCCCCAAGAGCGTAATCTGGAACATCGTATGGGTACATGGTGGCGGCG-3 (encoding the HA/His tag), were annealed and ligated to BamHI and EcoRI cutted pLenti-m1 vector, creating pLenti-m2. Free soluble C terminus of mouse sortilin was amplified using two primers 5-ACTACTACGCTCGAGAAGAAATATGTCTGTGGCGGAA-3 and 5-TCGAGTACGCTGCAGCTATTCCAGGAGGTCCTCATC-3. This fragment was cut with XhoI and PstI and ligated into the corresponding sites of pLenti-m2, creating pLenti-m2-HA/His-sSorC.

Supplemental References

Bogan, J.S., McKee, A.E., and Lodish, H.F. (2001). Insulin-responsive compartments containing Glut4 in 3T3–L1 and CHO cells: regulation by amino acid concentrations. Mol. Cell. Biol. 21, 4785–4806.

Supplemental Figure S1. myc7-Glut4 Is Faithfully Targeted in Differentiated Adipocytes

The 16,000g supernatant (800 µg total protein) from differentiated G cells and control empty vector (EV) transfected cells was fractionated by sucrose velocity centrifugation as described in Experimental Procedures. Odd fractions along with the pellet (P) of the gradient centrifugation were analyzed by Western blotting with the anti-myc antibody (top) or with the cocktail of anti-myc and 1F8 monoclonal antibodies (bottom). The dotted arrow indicates the direction of sedimentation.

Supplemental Figure S2. myc7-Glut4 Is Localized in Endosomes in Undifferentiated 3T3-L1 Cells

Undifferentiated G cells were incubated with Alexa-488-conjugated transferrin for 15 min and analyzed by immunofluorescence staining with anti-myc monoclonal antibody and Cy3-conjugated donkey anti-mouse IgG (Jackson Immunoresearch).

Supplemental Figure S3. Sortilin-myc/His Is Faithfully Targeted in Differentiated Adipocytes

(A) The 16,000g supernatant of differentiated S+ cells was immunoadsorbed with 1F8, anti-myc, and nonspecific IgG beads. An aliquot (100 µg) of postadsorptive supernatant along with Triton and SDS eluates were analyzed by Western blotting with antibodies against myc (top), sortilin (middle), and 1F8 (bottom).

(B) Light microsomes (800 µg of total protein) isolated from differentiated S+ cells treated and not treated with 100 nM insulin for 5 min were fractionated in a sucrose velocity gradient as described in Experimental Procedures. Western blot was stained with the cocktail of anti-myc and 1F8 monoclonal antibodies (top), anti-sortilin antibody (middle), and anti-cellugyrin antibody (bottom).

Supplemental Figure S4. Endogenous Glut4 Is Enriched in the Vesicular Fraction of S+ Cells

The 16,000g supernatant from differentiated S+ cells and control empty vector (EV)-transfected cells was pelleted by centrifugation at 200,000g for 90 min, and 50 µg of the resuspended pellet was analyzed by Western blotting.

Supplemental Figure S5. Correlation between Expression Levels of sortilin-myc/His and myc7-Glut4 in Individual Clones of GS Cells

Total cell lysates (100 µg of protein) were analyzed by Western blotting with the anti-myc antibody on the same membrane and rearranged in silico in the descending order. -actin is shown as a loading control.

Supplemental Figure S6. Sortilin Potentiates Insulin Responsiveness of Ectopically Expressed Glut4 in Swiss and NIH-3T3 Fibroblasts

Swiss and NIH-3T3 cells were transiently transfected with empty vectors (pBabe-puro and mLNCX2-EGFP), myc7-Glut4 (pBabe-myc7-Glut4 and mLNCX2), and myc7-Glut4 together with sortilin-myc/His (pBabe-myc7-Glut4 and mLNCX2-sortilin-myc/His), and 3H-2-deoxyglucose uptake was measured in transfected cells. The panel shows normalized mean values  SE of three independent measurements, each in duplicate.

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