PMC

(A) Schematic representation of Luman, with observed molecular weights of products. Arrowheads, possible glycosylation sites; HCF, HCF binding domain; DBD, DNA binding domain; TM, transmembrane domain, rxxl, putative S1P recognition motif; Acidic, acidic activation domain. The molecular weights of Luman and its fragments were determined by comparing their electrophoretic mobilities with those of protein molecular weight markers electrophoresed on the same gel. (B) Comparison of amino acid sequences of the transmembrane regions of human SREBP-2, ATF6, and Luman. Potentially important amino acids in Luman and the sites for the S221Op, R252A, R264G, and R267G mutations are boldfaced and numbered. Downward arrows point to the S1P and S2P cleavage sites for SREBP-2, and the LXXXXLXXXP sequence in the transmembrane domains of the three proteins is indicated.

A construct expressing the amino-terminal portion of Luman. (A) Plasmids specifying Lu or ΔTm, and two independent clones in which the codon for serine 221, which lies just amino-terminal to the transmembrane domain, was replaced with an Opal codon (S221O1 and S221O2), were expressed in the TnT in vitro transcription-translation system and analyzed on an SDS-10% polyacrylamide gel. (B) Detection of Luman and Luman S221Op in transfected cells by immunofluorescence. Note the cytoplasmic location of Luman and the nuclear location of S221Op as well as the relatively few cells that show detectable S221Op. Panel 3 shows S221Op-expressing cells treated with the proteosomal inhibitor MG132. (C) Immunoblots of Luman, Luman ΔTm, and Luman S221Op expressed in Vero cells with or without MG132, separated on an SDS-8% polyacrylamide gel. The arrowhead and arrow on the left indicate full-length Luman and the potential amino-terminal cleaved product of Luman, respectively. The arrow on the right indicates S221Op. Note that relatively little S221Op was detected unless proteosomal digestion was suppressed by MG132.

Effect of brefeldin A on Luman. (A) Detection of brefeldin A-generated fragments in the nucleus. Cells were transfected with pcFL-Lu[ΔTM] (ΔTM) or pcFL-Lu-HA (Lu). All cultures were treated with MG132 to suppress degradation of the activated amino-terminal Luman fragment. One culture of cells transfected with pcFL-Lu-HA was also treated with brefeldin A (Lu + bref) from 12 h after transfection. Forty hours after transfection, cells were harvested and fractionated to collect the nuclear fraction, and whole and nuclear fractions were separated on an SDS-12% polyacrylamide gel and immunoblotted with antibodies against Luman or HA. Open arrowhead in lane 3, 40K polypeptide; solid arrowhead in lanes 3 and 6, 35K polypeptide; solid arrowhead in lane 9, ∼20K fragment. (B) Cells were treated as described in the legend to panel A and immunoblotted with antibodies against calnexin (arrowhead). (C) Cultures were transfected with pcFL-Lu-HA either alone or with pS1P-KDEL or pS1P-KDAS. Cultures transfected with pcFL-Lu-HA were either left untreated or treated with brefeldin A. Lysates were electrophoresed on SDS-10% polyacrylamide gels and immunoblotted with an anti-Luman serum or an anti-HA or anti-Myc monoclonal antibody. Open arrowhead, S1PA; solid arrowhead, S1PB and S1PC ().

Effect of brefeldin A on Luman. (A) COS cells transfected with 2 μg of expression vectors for Luman (lanes 1 to 7) (pJS6) or Luman ΔTM (lanes 8 to 14) (pJS7). At 24 h posttransfection, the medium was replaced with Dulbecco's modified Eagle's medium containing 1 μg of brefeldin A(brf)/ml, and cells were incubated for the times indicated. Total-cell extracts were subjected to SDS-polyacrylamide gel electrophoresis and immunoblot analysis using an SV5 monoclonal antibody (dilution, 1/10,000) that recognized the extreme amino termini of the Luman proteins. Arrowhead indicates the 40K polypeptide. (B) COS cells grown on coverslips were transfected with pJS6. At 24 h posttransfection, cells were treated with 1 μg of brefeldin A/ml alone or with 10 μM MG132, as indicated. Cells were fixed in methanol and examined by confocal microscopy. Arrowheads indicate sites of Luman accumulation in the nucleus.

Luman is posttranslationally modified by glycosylation. (A) Immunoblot of Luman from transfected Vero cells (lane 1) and autoradiograph of the in vitro TnT system (Promega) charged with pcDNA3 (lane 2) or pcFL-Lu (lane 3). Cell and TnT lysates were separated on an SDS-8% polyacrylamide gel and blotted. Strips were then analyzed by immunoblotting or by autoradiography. (B) Immunoblot analysis of Luman-expressing cells after incubation in the presence of tunicamycin. Vero cells in six-well plates were transfected with pcFL-Lu. After 24 h, the medium was replaced with fresh medium containing tunicamycin at a concentration of 2 μg/ml. At the indicated times, cells were lysed, separated on an SDS-10% polyacrylamide gel, and analyzed by immunoblotting with an anti-Luman antibody. (C) Luman is glycosylated. Lysates of Luman-expressing cells treated with tunicamycin (Tun) or membranes from fractionated Luman-expressing cells that were either left untreated (pLu) or digested with Endo H or PNGase F were separated on an SDS-10% polyacrylamide gel and analyzed by immunoblotting. Lanes represent lysates from equivalent numbers of cells. The positions of protein molecular weight markers (in thousands) electrophoresed on the same gels are indicated on the left. The arrowhead points to Luman, while the arrow indicates unglycosylated (lane 2) or deglycosylated (lanes 3 and 4) Luman. The 61K band present in all lanes is a cellular protein that reacts nonspecifically to the anti-Luman serum.

Effects of mutation of arginines 252, 264, and 267 at the putative S1P cleavage sites on generation of the amino-terminal cleaved fragment of Luman. (A) Vero cells were transfected with plasmids expressing either FL-Lu-HA (Luman) or FL-Lu-HA(R252A) expressed from the CMV IE promoter or an HSV TK promoter. The CMV IE and HSV TK promoters containing blank plasmids, pcDNA3 and pTK3, were used as controls. Cells were cotransfected with pBB15, a reporter plasmid with coding sequences for CAT linked to the basal HSV LAT and pCMV-β-Gal. CAT activity is adjusted for transfection efficiency. (Inset) Western blot of lysates of cells transfected with the pTK constructs. Arrow indicates the location of bands representing Luman and Luman(R252A). The lower-molecular-weight band in all lanes is due to nonspecific binding of the anti-Luman antibody. (B) Lysates of cells expressing LuFL-Lu-HA(R252A), either left untreated (Lu), treated with brefeldin A (Lu + bref), or cotransfected with either pS1P-KDEL or pS1P-KDAS, were separated on SDS-10% polyacrylamide gels and probed with anti Luman serum. (C) Cells transfected with pBB15 and (from left to right) either pcDNA, pcFL-Lu-HA (Luman), pcFL-Lu-HA(R264G), pTK3, pTKFL-Lu-HA (Luman), pTKFL-Lu-HA(R264G), or pTKFL-Lu-HA(R267G) were assayed for CAT activity. (Inset) Western blot of lysates of cells transfected with the pTK constructs. (D) Cells transfected with plasmids expressing Luman or its mutants (R252A, R264G, or R267G), either alone or in conjunction with plasmids expressing either S1PKDEL or S1PKDAS, were analyzed by immunoblotting. Cells expressing Luman or mutant proteins alone were either left untreated (lanes 1, 5, 9, and 13) or treated with brefeldin A (lanes 2, 6, 11, and 14).