CASP, the alternatively spliced product of the gene encoding the CCAAT-displacement protein transcription factor, is a Golgi membrane protein related to giantin

Abstract

Large coiled-coil proteins are being found in increasing numbers on the membranes of the Golgi apparatus and have been proposed to function in tethering of transport vesicles and in the organization of the Golgi stack. Members of one class of Golgi coiled-coil protein, comprising giantin and golgin-84, are anchored to the bilayer by a single C-terminal transmembrane domain (TMD). In this article, we report the characterization of another mammalian coiled-coil protein, CASP, that was originally identified as an alternatively spliced product of the CUTL1 gene that encodes CCAAT-displacement protein (CDP), the human homologue of the Drosophila homeodomain protein Cut. We find that the Caenorhabditis elegans homologues of CDP and CASP are also generated from a single gene. CASP lacks the DNA binding motifs of CDP and was previously reported to be a nuclear protein. Herein, we show that it is in fact a Golgi protein with a C-terminal TMD and shares with giantin and golgin-84 a conserved histidine in its TMD. However, unlike these proteins, CASP has a homologue in Saccharomyces cerevisiae, which we call COY1. Deletion of COY1 does not affect viability, but strikingly restores normal growth to cells lacking the Golgi soluble N-ethylmaleimide-sensitive factor attachment protein receptor Gos1p. The conserved histidine is necessary for Coy1p's activity in cells lacking Gos1p, suggesting that the TMD of these transmembrane Golgi coiled-coil proteins is directly involved in their function.

Figure 1

A family of proteins related to CASP, the alternatively spliced product of the CUTL1 gene. (A) Diagrammatic representation of human CDP, CASP, and the product of the yeast ORF YKL179c. CDP and CASP share an N-terminal region, with the point of divergence indicated by an arrow. The cut repeat and homeobox (Hx) domains in CDP are shown, along with coiled-coil predictions for the proteins (Lupas et al., 1991). (B) Alignment of the C termini of homologues of CASP, golgin-84, and giantin from the indicated species. The putative TMD is boxed and the conserved histidine and tyrosine residues indicated by triangles. The CASP homologues are as in C, plus Candida albicans orf6.3753; S. pombe SPCC364.04c; Neurospora crassa 9G6.340. The giantin homologues are from ESTs, GenBank accession numbers BM486947 and BJ037357. (C) Species distribution of homologues of the indicated proteins, with the gene names given, and the number of residues in brackets. Cux-2 is a neuronal-specific isoform of CDP found in humans and mice that does not seem to have an alternative product analogous to CASP. (D) Structure of two C. elegans cDNAs from the region of the predicted genes Y54F10AM.4 and Y54F10AM.3 (cDNAs kindly provided by Yuji Kohara, National Institute of Genetics, Mishima, Japan). The positions in the genome of the exons present in the cDNAs are indicated. The cDNAs share exons but then diverge to encode the C. elegans homologues of CDP and CASP.

Figure 2

CASP behaves as a type II integral membrane protein of the Golgi apparatus. (A) Immunofluorescence localization of CASP in normal rat kidney fibroblasts. Cells fixed in 4% paraformaldehyde were permeabilized by freeze-thaw and either treated with 0.5% Triton X-100 in PBS (top) or left untreated (bottom). Cells were probed with antibodies against CASP and the Golgi protein TGN38 as described in MATERIALS AND METHODS, with the omission of Tween 20. (B) Anti-CASP protein blot of rat liver Golgi membranes (5 μg/lane) solubilized in SDS sample buffer with or without 5% β-mercaptoethanol (β-ME). (C) Coomassie Blue-stained protein gel of immunoprecipitates from rat liver Golgi membranes solubilized in digitonin, by using anti-CASP or a control IgG. Proteins identified by mass spectrometry are indicated. (D) Anti-CASP protein blot of rat liver Golgi membranes extracted with PBS containing either nothing (PBS), 1.5 M KCl, 100 mM sodium carbonate, pH 11.5 or 1% Triton X-100. Membranes (1 mg/ml) were extracted for 30 min at 4°C, centrifuged (105,000 × g for 40 min), and equivalent amounts of pellet (P) and supernatant (S) were analyzed by SDS-PAGE and protein blotting.

Figure 3

Redistribution of CASP after treatment with brefeldin A (BFA). Immunofluorescence micrographs of COS cells treated with 10 μg/ml BFA at 37°C for the times indicated, and then fixed, permeabilized, and stained with antibodies against CASP and β′-COP. In the right-hand panels cells were treated for 45 min with BFA, washed thoroughly, and allowed to recover for 60 min.

Figure 4

The C terminus of CASP contains a TMD important for Golgi targeting. Confocal micrographs of COS cells expressing GFP fusions to the C-terminal 160 residues of CASP [GFP-CASP(519–678)] (A), or full-length CASP (GFP-CASP), or to CASP with a TMD mutation [GFP-CASP(Y624L)] (B). In A, the cells were fixed, permeabilized, and stained with antibodies against the Golgi marker protein giantin.

Figure 5

Subcellular localizations of CDP and CASP are distinct. Confocal micrographs of COS cells expressing either full-length CDP fused to GFP (CDP-GFP), or the N-terminal 749 residues fused to GFP [CDP(1–749)-GFP]. Cells were also stained with antibodies to endogenous CASP or TGN46.

Figure 6

Yeast ORF YKL179c is expressed under normal growth conditions and encodes a protein that localizes to the early Golgi. (A) Anti-HA protein blots of total protein from yeast strain APY02 in which the YKL179c ORF is followed by a triple HA epitope tag (YKL179c-3xHA) or from the parental strain SEY6210 (con.). (B) Confocal micrographs of APY02 double labeled with anti-HA monoclonal 12CA5 and antibodies against either the early Golgi protein Anp1p or the late Golgi protein Tlg1p.

Figure 7

Genetic interactions between COY1/YKL179c and the SNARE-encoding genes GOS1 and SEC22. (A) Tetrad analysis of spores from a gos1Δ::kanMx/GOS1, ykl179cΔ::HIS5/YKL179c diploid strain. Tetrads were dissected and incubated on YEPD plates at 30°C for 3 d (complete), and markers were then tested by replica plating on kanamycin (+ Kan) and minus-histidine plates (− His). Four representative tetrads are shown. The small segregants in each tetrad are Kan^res, but segregants that are both Kan^res and HIS⁺ do not display any growth or germination defect. (B) Tetrad analysis as in A of spores from a sec22Δ::kanMx/SEC22, ykl179cΔ::HIS5/ YKL179cdiploid strain (AGY03). Segregants that contained sec22Δ::kanMx alone showed a small but reproducible growth defect, which was not seen in those that also contained ykl179cΔ::HIS5. (C) Growth at the indicated temperatures of the temperature-sensitive sec22-3 strain containing the 2 μ plasmid pRS426 with (+) or without (−) full-length Coy1p expressed from a constitutive PHO5 promoter. Ten-fold serial dilutions of cells were spotted onto selective plates and incubated at the indicated temperatures for 3 d. (D) Growth of parental strain SEY6210 (WT) or gos1-Δ2 containing 2 μ plasmids as in C.

Figure 8

Role of the conserved TMD residues in the Coy1p function. (A) Anti-HA protein blots of total protein from yeast expressing HA-tagged Coy1p (wt) or the same protein with the indicated TMD mutations. Proteins were expressed in strain gos1-Δ1 from CEN plasmids under the control of the galactose-regulated GAL1 promoter and induced with 2% galactose for 3 h at 30°C. (B) Confocal micrographs of yeast as in A. After induction as in B, cells were fixed, permeabilized, and stained with antibodies against the HA epitope and the Golgi marker Anp1p. (C) Effect of TMD mutants on the ability of Coy1p to perturb growth of the gos1-Δ1 strain. Cells were transformed with plasmids as in A but expressing Coy1p (wt) without an epitope tag, or the same protein with the indicated TMD mutants, or no gene (−). Yeast were grown overnight in media containing 2% raffinose, spotted onto selective plates containing either 2% glucose or 2% galactose, and incubated at the indicated temperatures for 2–3 d.