Subunit structure of the mammalian exocyst complex

Abstract

The exocyst is a protein complex required for the late stages of secretion in yeast. Unlike the SNAREs (SNAP receptors), important secretory proteins that are broadly distributed on the target membrane, the exocyst is specifically located at sites of vesicle fusion. We have isolated cDNAs encoding the rexo70, rsec5, and rsec15 subunits of the mammalian complex. The amino acid sequences encoded by these genes are between 21% and 24% identical to their yeast homologs. All three genes are broadly expressed and multiple transcripts are observed for rexo70 and rsec15. Characterization of cDNAs encoding the 84-kDa subunit of the mammalian complex revealed a novel protein. mAbs were generated to the mammalian rsec6 subunit of the exocyst complex. rsec6 immunoreactivity is found in a punctate distribution at terminals of PC12 cell processes at or near sites of granule exocytosis.

Figure 1

Subunits of the mammalian exocyst complex. The purified mammalian exocyst complex is comprised of eight bands ranging in size from 71 to 110 kDa. Previous studies defined three subunits as rsec10, rsec6, and rsec8. In this report we characterize rexo70, rsec15, and rsec5 and a set of cDNAs that predicts a protein containing peptides derived from the 84-kDa band.

Figure 2

Comparisons of the predicted amino acid sequences of mammalian exocyst subunits with respective Caenorhabditis elegans and yeast homologs. Predicted amino acid sequences of rexo70, rsec15, and rsec5 derived from rat cDNA analyses are compared with the homologous sequences in the database with the pileup program of GCG. Identical residues are in a black box with white letters and similar residues are shaded. Dotted regions represent gaps. Lines above the amino acid sequences indicate the peptides determined by amino acid sequencing of the subunits of the purified mammalian complex. (A) rexo70: Accession numbers of C. elegans and yeast homologs are U80437 and P19658. The smallest sum probability for the homology between the C. elegans and rat exo70 is 2.6 × 10⁻²⁴. pileup analysis indicates that rat and C. elegans share 25% identity and 42% similarity, rat and yeast share 19% identity and 34% similarity, and C. elegans and yeast share 17% identity and 30% similarity. (B) rsec15: Accession numbers of C. elegans and yeast homologs are U41026 and Z72755, respectively. The smallest sum probability for the homology between the C. elegans and rat exo70 is 2.2 × 10⁻¹⁵³. pileup analysis indicates that rat and C. elegans share 36% identity and 50% similarity, rat and yeast share 18% identity and 34% similarity, and C. elegans and yeast share 16% identity and 30% similarity. An asterisk (∗) at the end of the amino acid sequences of C. elegans homolog indicates the first possible stop codon in the genomic sequence. (C) rsec5: Accession numbers of C. elegans and yeast homologs are Z68319 and Z50046. The smallest sum probability for the homology between the C. elegans and rat exo70 is 3.2 × 10⁻⁶¹. pileup analysis indicates that rat and C. elegans share 28% identity and 46% similarity, rat and yeast share 18% identity and 31% similarity, and C. elegans and yeast share 13% identity and 27% similarity. (D) exo84: The 716 amino acid protein contains the sequences of seven peptides determined from amino acid sequencing of the 84-kDa band. The peptide sequences are underlined.

Figure 2

Comparisons of the predicted amino acid sequences of mammalian exocyst subunits with respective Caenorhabditis elegans and yeast homologs. Predicted amino acid sequences of rexo70, rsec15, and rsec5 derived from rat cDNA analyses are compared with the homologous sequences in the database with the pileup program of GCG. Identical residues are in a black box with white letters and similar residues are shaded. Dotted regions represent gaps. Lines above the amino acid sequences indicate the peptides determined by amino acid sequencing of the subunits of the purified mammalian complex. (A) rexo70: Accession numbers of C. elegans and yeast homologs are U80437 and P19658. The smallest sum probability for the homology between the C. elegans and rat exo70 is 2.6 × 10⁻²⁴. pileup analysis indicates that rat and C. elegans share 25% identity and 42% similarity, rat and yeast share 19% identity and 34% similarity, and C. elegans and yeast share 17% identity and 30% similarity. (B) rsec15: Accession numbers of C. elegans and yeast homologs are U41026 and Z72755, respectively. The smallest sum probability for the homology between the C. elegans and rat exo70 is 2.2 × 10⁻¹⁵³. pileup analysis indicates that rat and C. elegans share 36% identity and 50% similarity, rat and yeast share 18% identity and 34% similarity, and C. elegans and yeast share 16% identity and 30% similarity. An asterisk (∗) at the end of the amino acid sequences of C. elegans homolog indicates the first possible stop codon in the genomic sequence. (C) rsec5: Accession numbers of C. elegans and yeast homologs are Z68319 and Z50046. The smallest sum probability for the homology between the C. elegans and rat exo70 is 3.2 × 10⁻⁶¹. pileup analysis indicates that rat and C. elegans share 28% identity and 46% similarity, rat and yeast share 18% identity and 31% similarity, and C. elegans and yeast share 13% identity and 27% similarity. (D) exo84: The 716 amino acid protein contains the sequences of seven peptides determined from amino acid sequencing of the 84-kDa band. The peptide sequences are underlined.

Figure 2

Comparisons of the predicted amino acid sequences of mammalian exocyst subunits with respective Caenorhabditis elegans and yeast homologs. Predicted amino acid sequences of rexo70, rsec15, and rsec5 derived from rat cDNA analyses are compared with the homologous sequences in the database with the pileup program of GCG. Identical residues are in a black box with white letters and similar residues are shaded. Dotted regions represent gaps. Lines above the amino acid sequences indicate the peptides determined by amino acid sequencing of the subunits of the purified mammalian complex. (A) rexo70: Accession numbers of C. elegans and yeast homologs are U80437 and P19658. The smallest sum probability for the homology between the C. elegans and rat exo70 is 2.6 × 10⁻²⁴. pileup analysis indicates that rat and C. elegans share 25% identity and 42% similarity, rat and yeast share 19% identity and 34% similarity, and C. elegans and yeast share 17% identity and 30% similarity. (B) rsec15: Accession numbers of C. elegans and yeast homologs are U41026 and Z72755, respectively. The smallest sum probability for the homology between the C. elegans and rat exo70 is 2.2 × 10⁻¹⁵³. pileup analysis indicates that rat and C. elegans share 36% identity and 50% similarity, rat and yeast share 18% identity and 34% similarity, and C. elegans and yeast share 16% identity and 30% similarity. An asterisk (∗) at the end of the amino acid sequences of C. elegans homolog indicates the first possible stop codon in the genomic sequence. (C) rsec5: Accession numbers of C. elegans and yeast homologs are Z68319 and Z50046. The smallest sum probability for the homology between the C. elegans and rat exo70 is 3.2 × 10⁻⁶¹. pileup analysis indicates that rat and C. elegans share 28% identity and 46% similarity, rat and yeast share 18% identity and 31% similarity, and C. elegans and yeast share 13% identity and 27% similarity. (D) exo84: The 716 amino acid protein contains the sequences of seven peptides determined from amino acid sequencing of the 84-kDa band. The peptide sequences are underlined.

Figure 3

Multiple tissue Northern blot analysis. Size markers are on the left in kb. (A) rexo70: Two transcripts of 2.3 and 3.1 kb are observed all tissues examined. (B) rsec15: Two transcripts of 2.7 and 3.5 kb are observed in all tissues. (C) rsec5: A major transcript of 5.2 kb is observed in all tissues.

Figure 4

Tissue distribution of rsec6 protein. (A) Western blot detection of rsec6 in brain postnuclear supernatant by anti-rsec6 mAbs 9E9 and 9H5. (B) Regional Western blot analysis of rsec6 using anti-rsec6 mAb 9H5. Samples (20 μg) of postnuclear supernatant proteins from the indicated tissues were loaded per lane.

Figure 5

rsec6 colocalizes with vesicles and sites of exocytosis in nerve growth factor-differentiated PC12 cells. (A and B) Comparison of the distribution of rsec6 and the synaptic vesicle marker, synaptotagmin, in PC12 cells that have been differentiated with nerve growth factor for 2 days demonstrated by double immunofluorescence. The antibodies used are listed above their respective fluorescence micrographs. Differential interference contrast micrographs of each cell are shown on the left. A shows that rsec6 immunofluorescence is more intense in the vesicle-rich terminals of growing neurites with lighter labeling in the cell body. (B) A higher magnification view reveals the punctate distribution of rsec6 in the neurite terminal. (C) rsec6 is also found at sites of Ca²⁺-regulated secretion as demonstrated by the presence of DβH on the cell surface. Antibodies directed against DβH were applied in the absence of detergent to reveal cell-surface sites of recent exocytosis in response to K⁺/Ca²⁺-induced depolarization. Subsequent application of antibodies to rsec6 in the presence of detergent reveal the distribution of internal rsec6. The overlay micrograph demonstrates the contrast between intracellular rsec6 localization and cell-surface DβH distribution. Bar (in C) = 7.8 μm (A), 3.3 μm (B), 6.8 μm (C).