Identification of the putative mammalian orthologue of Sec31P, a component of the COPII coat

Abstract

The regulation of intracellular vesicular trafficking is mediated by specific families of proteins that are involved in vesicular budding, translocation, and fusion with target membranes. We purified a vesicle-associated protein from hepatic microsomes using sequential column chromatography and partially sequenced it. Oliogonucleotides based on these sequences were used to clone the protein from a rat liver cDNA library. The clone encoded a novel protein with a predicted mass of 137 kDa (p137). The protein had an N terminus WD repeat motif with significant homology to Sec31p, a member of the yeast COPII coat that complexes with Sec13p. We found that p137 interacted with mammalian Sec13p using several approaches: co-elution through sequential column chromatography, co-immunoprecipitation from intact cells, and yeast two-hybrid analysis. Morphologically, the p137 protein was localized to small punctate structures in the cytoplasm of multiple cultured cell lines. When Sec13p was transfected into these cells, it demonstrated considerable overlap with p137. This overlap was maintained through several pharmacological manipulations. The p137 compartment also demonstrated partial overlap with ts045-VSVG protein when infected cells were incubated at 15 degrees C. These findings suggest that p137 is the mammalian orthologue of Sec31p.

Fig. 1

Purification and cloning of p137. (A) SDS-PAGE (8% acrylamide) of the peak fraction from the S300 gel chromatography step: (1) Coomassie Blue; (2) immunoblot with antibody 212. The protein band corresponding to 170 kDa was subjected to partial amino acid sequencing. (B) Cloning strategy used to obtain the complete p137 cDNA. The coding region spans 3786 nucleotides, not including the stop codon (hatched box). The 5′ and 3′ untranslated regions include an additional 672 nucleotides (solid boxes). The locations of the six peptides obtained from the purified protein are indicated by the solid bars numbered 1–6. The two probes used to screen the rat liver cDNA library are indicated by solid bars labeled ‘1st probe’ and ‘2nd probe’. Clones 1A, 2A, and 9A were obtained from the initial screen of a rat liver cDNA library and clones 2B, 11A, and 18B from the second screen of the same library. The small hatched box indicates the 5′ RACE product. (C) cDNA and amino acid sequence of p137. Shown is the nucleotide sequence of the combination of overlapping clones that encompass the coding region and the adjacent 5′- and 3′-noncoding regions, with 1 representing the first nucleotide of the coding region. The deduced amino acid sequence of p137 is shown above the nucleotide sequence. Nucleotide sequences encoding the six peptides obtained from the purified protein are underlined. The sequence of the fusion protein used for antibody production is indicated with a dash underline. A possible polyadenylation signal sequence (ATTAAA) is shown in bold. These sequence data are available from GenBank/EMBL/DDBJ under accession number AFO34582.

Fig. 2

Anti-p137 antibodies detect a 170 kDa protein in rat liver, WIF-B cells, NRK cells and isolated yeast Sec13/13p complexes that comigrates with purified p137. Purified p137 (lane 1) and total homogenates of rat liver (lane 2, 20 μg), WIF-B cells (lane 3, 20 μg), and NRK cells (lane 4, 20 μg) were separated by SDS-PAGE (8–12% acrylamide) and subjected to immunoblot analysis using mouse anti-p137 (A) or rabbit anti-p137 (B) antibodies. Both antibodies detect a 170 kDa band that comigrates with purified p137. Purified yeast Sec13/31p complex and p137 were subjected to immunoblot analysis using rabbit anti-p137 (C). The antibody recognizes both p137 and yeast Sec31p (arrowhead), but not yeast Sec13p (arrow); the two additional bands detected are Sec31p degradation products (asterisk).

Fig. 3

Domain structure of p137 protein and homology to Sec31p. (A) The deduced protein sequence of p137 can be divided into four domains: a WD-repeat region (I), an intervening region (II), a proline-rich region (III) and a C-terminal region (IV). (B) Percentage identity of p137 with Sec31p from S. pombe and S. cerevisiae. (C) Comparison of the WD-repeat region of rat liver p137 to KIAA0905 (human), S. pombe and S. cerevisiae Sec31p. Conserved amino acid sequences are shaded. WD domains are boxed.

Fig. 4

p137 mRNA expression. p137 mRNA expression was analyzed in a human organ northern blot. Sizes of the RNA markers (kb) are indicated on the left. Message encoding p137 is expressed as a major 4.3 kb band.

Fig. 5

Co-elution of p137 and Sec13p by column chromatography. The supernatant of the microsomal fraction was applied sequentially to hydroxyapatite, Sephacryl S300, and MonoQ HR 5/5 columns. The eluted fractions were solubilized and separated on SDS-PAGE (8% acrylamide), then subjected to immunoblot analysis using antibody 212 (to detect p137) and anti-human Sec13 antibody. The elution profiles for both protein bands coincide at each chromatography step.

Fig. 6

Anti-p137 co-immunoprecipitates Sec13p. Non-ionic detergent extracts prepared from a rat liver S100 fraction were immunoprecipitated with the rabbit anti-p137 (third lane), non-immune IgG (second lane) or no antibody (first lane), separated on SDS-PAGE (8 to 12% acrylamide) and transferred to Immobilon. The blot was probed with anti-p137 (top panel) and anti-human Sec13p (bottom panel). Single bands at the appropriate mobilities were detected only when specific antibody to p137 was used in the immunoprecipitation step.

Fig. 7

p137 interacts with mammalian Sec13p and Sec23p but not yeast Sec13p and Sec23p in a yeast two-hybrid assay. (A–B) Two hybrid interactions among Sec31p, p137, Sec13p and Sec23p were detected by β-galactosidase assay. (C) Gene fragments of Sec31 and p137 are indicated in the diagrams and by the amino acid numbers included in parentheses. nd, not done.

Fig. 8

The vesicular compartment containing p137 colocalizes with transfected Sec13p. Clone 9 cells (top row) or NRK cells (bottom row) were transfected with Sec13p-GFP or Sec13p with a T7 tag, respectively, fixed, then incubated with anti-p137 alone (Clone 9) or together with anti-T7 antibody (NRK). Sec13p was localized to cytoplasmic vesicles in both cell lines; it was also found over the nucleus in some cells. p137 was distributed to small cytoplasmic vesicles, but not over the nucleus. Most of the vesicular labeling for p137 and Sec13p overlapped (merged images). Bars, 10 μm.

Fig. 9

p137 and Sec13p remain associated during different experimental treatments. (A–C) Cells transfected with T7-Sec13p plasmid were treated with 10 μg/mL brefeldin A (BFA) for 1 hour at 37°C, then fixed and labeled with mouse anti-p137 antibody (A, p137) and anti-T7 antibody (B, Sec13p). This treatment redistributed the Golgi protein mannosidase II into the ER (not shown). In the presence of brefeldin A, p137 and Sec13p remain co-localized and associated with vesicles (C, merge). (D–F) Cell transfected with T7-Sec13p plasmid were treated with 33 μM nocodazole for 1 h, then fixed and labeled with the same antibodies as described in A. This treatment depolymerized microtubules, as demonstrated by staining with anti-tubulin antibody (not shown); however, p137 (D) and Sec13p (E) remained co-localized (F) but became more widely distributed in the cytoplasm than in control cells.

Fig. 10

p137 colocalizes with a marker of ER-derived transport vesicles. NRK cells were transfected at the restrictive temperature (40°C) with a plasmid encoding tsO45 VSVG. The cells were kept at 40°C (top panel) or shifted to either 32°C (middle panel) or 15°C (bottom panel) for 1 hour. The cells were then fixed and labeled with mouse anti-p137 (A,D,G) and rabbit anti-VSVG (B,E,H). As shown in the merged images (C,F,I) at 15°C but not at 40°C or 32°C, p137 overlaps with VSVG in pre-Golgi structures. In the merged image: green, VSVG; red, p137; yellow, areas of colocalization. Arrows in G and H indicate an area of colocalization that is also shown in the magnified inset (I).