Mutational analysis of betaCOP (Sec26p) identifies an appendage domain critical for function

Abstract

Background: The appendage domain of the gammaCOP subunit of the COPI vesicle coat bears a striking structural resemblance to adaptin-family appendages despite limited primary sequence homology. Both the gammaCOP appendage domain and an equivalent region on betaCOP contain the FxxxW motif; the conservation of this motif suggested the existence of a functional appendage domain in betaCOP.

Results: Sequence comparisons in combination with structural prediction tools show that the fold of the COOH-terminus of Sec26p is strongly predicted to closely mimic that of adaptin-family appendages. Deletion of the appendage domain of Sec26p results in inviability in yeast, over-expression of the deletion construct is dominant negative and mutagenesis of this region identifies residues critical for function. The ArfGAP Glo3p was identified via suppression screening as a potential downstream modulator of Sec26p in a manner that is independent of the GAP activity of Glo3p but requires the presence of the COOH-terminal ISS motifs.

Conclusion: Together, these results indicate an essential function for the predicted betaCOP appendage and suggest that both COPI appendages perform a biologically active regulatory role with a structure related to adaptin-family appendage domains.

Figure 1

Alignment of COPI and AP2 appendage domains. A structure-based sequence alignment of the appendage domains of α- and βAP2 with that of γCOP was generated as described in [16]. Secondary structural elements of the γCOP and the AP2 appendage domains determined previously are indicated above the sequence alignment. The blue arrow denotes the position of the boundary between the platform and the Ig-like (β-sandwich) subdomains. Highlighted in red is where four or more sequences contained a strictly conserved residue at that position. Regions of similarity, calculated using functional amino acid groupings, are boxed in blue. Residues for which no structural information is available were positioned using a primary sequence alignment using the ClustalX 1.83 program and manual inspection. The conserved FxxxW motif is indicated below the sequence alignment in pink. The predicted secondary structural elements for the appendage domain of βCOP, Sec26p are shown beneath the alignment as determined with the PSIPRED confidence level of the assignments shown in the diagram.

Figure 2

Functional importance of the βCOP appendage domain. A. Schematic illustrating the characteristics of the various Sec26p truncation constructs used for experiments. B. Plasmids coding for truncated constructs of SEC26, wild type SEC26, or vector only were transformed into the sec26Δ tester strain and spotted onto complete media (YPD) and synthetic complete media (SCD) with 5-FOA at 25, 30, 34, 37, and 40°C. C. Plasmids coding for vector alone or full length or truncated constructs under control of the copper-inducible promoter (P_CUP1) were transformed into wild type yeast and replica-plated onto minimal media (SD) with and without the addition of Cu²⁺ (0 or 0.3 mM CuSO₄). Growth was followed for three or seven days (3d and 7d respectively). The dominant negative yip1-6 construct was included as a negative control [71]. D. Transformed cells were grown in liquid minimal media with and without the exogenous CuSO₄. Cultures were grown to early log phase and adjusted to an OD₆₀₀ of 0.2. CuSO₄ was added at the levels indicated and cells were grown at 30°C with turbidity measured every half hour for 2 days.

Figure 3

Sec26p mutant allele creation. A. Bar chart showing the relative location and number of each mutated amino acid position identified in the ts allele set created by random mutagenesis. Superimposed is the % identity of each residue derived from a sequence alignment of βCOP sequences in Clustal v1.83. For reference, a grid indicates the positions of amino acid residues that are aligned (according to Figure 1) with solvent exposed residues of γCOP, located within 3Å distance of the FxxxW motif, and identified as a minimal set of functionally important residues through GLO3 interactions. B. Expression of constructs revealed with anti-Sec26p appendage domain antibody. Whole cell lysates were prepared from (Lane 1) wild type cells expressing endogenous Sec26p (109 kDa), (Lane 2) cells expressing GFP-Sec26p as the only copy of Sec26p (136 kDa, pRC2798), (Lane 3) cells over-expressing Sec26p under control of the copper-inducible promoter (P_CUP1) (42.2 kDa, pRC2630a), or (Lane 4) cells expressing both wild type levels of Sec26p plus over-expressed Sec26p appendage under control of the copper-inducible promoter (P_CUP1) (pRC2629b). C. Invertase secretion in sec26^ts mutant cells. Several mutant alleles were chosen and invertase secretion was measured after a one hour shift to the restrictive temperature. The graph shows % secretion relative to wild type and an alternative COPI ts mutant, the deletion of sec28 (εCOP subunit). Data plotted is the average of three experiments and confidence intervals shown with error bars. D. Sec26p allele expression levels. Cellular lysates prepared from each of the sec26^ts strains after incubation at 40°C for 1 h to assess Sec26p expression levels at the restrictive temperature. Blots were subsequently probed with an anti-Elp1p (~150 kDa) antibody [70] as a loading control.

Figure 4

Phenotype of sec26^ts Mutants. A. Schematic of strategy to create appendage domain mutants for suppressor screening. ts alleles created as described in Materials and Methods were subcloned into the vector pRS305 for one-step gene disruption and replacement to create integrated genomic sec26 alleles. B. The sec26Δ tester strain was transformed with each of the sec26^ts mutant plasmids and plated on synthetic complete media (SCD) with 5-FOA to counter-select for the wild type SEC26 plasmid (not shown). The resultant strains were plated at 30, 34, 37, and 40°C to demonstrate the temperature sensitive phenotype of each mutant (top row). Each strain was transformed with a GLO3 expressing plasmid under control of the galactose-inducible promoter (P_GAL1/10) and plated on SCG at the above listed temperatures to evaluate the ability of GLO3 to suppress the ts phenotype (bottom row). The data is also summarized in Table 1. C. The sec26Δglo3Δ tester strain was transformed with each of the sec26^ts mutant plasmids and streaked onto SCD + 5-FOA to assess growth when sec26^ts was expressed as the only copy of SEC26 in the glo3Δ background. One representative plate is shown. Data for all sec26 alleles is summarized in Table 1. D. Diagram showing the numbers of residues identified at the intersection of at least three of the Groups I-IV together with the residue number in Sec26p. E. Mapping of residues identified in Figure 4D onto the known structure of γCOP appendage domain. The γCOP appendage structure is depicted as a ribbon diagram with α-helices in red and β-strands in yellow. The calculated molecular surface (transparent grey) is overlaid on the ribbon diagrams. Selected residues (K766, T759, A638, D867) were identified from the alignment shown in Figure 1 as the equivalent of the Sec26p residues chosen as described in Figure 4D and shown in blue. For clarity, the residues surrounding and including the FxxxW motif are shown in additional colors: pink (F772), light blue (W776), green (A775), and orange (V779). The appendage is shown from a "side" view (center image) with the NH₂-terminal α-helix projecting toward the viewer. The image was then rotated 90° for the "front" view (left image), 90° for the "back" view (right image), and 90° towards the viewer for the "top" view (top image). Each of the selected residues has surface exposure.

Figure 5

Glo3p-GFP localization in the presence of over-expressed Sec26p appendage. A. Functionality and localization of Glo3p-GFP. The functionality of the COOH-terminally tagged Glo3p-GFP construct (pRC3341A) was checked by transformation into the heterozygous diploid (MATa/α his3Δ0/his3Δ0 leu2Δ0/leu2Δ0 ura3Δ0/ura3Δ0 MET15/met15Δ0 LYS2/lys2Δ0 GLO3/glo3Δ::HIS3 GCS1/gcs1Δ::KAN^R). Following sporulation, haploid strains deleted for the genomic copies of GLO3 and GCS1, were identified. Viable haploids that were His⁺ and KAN^R were also positive for Glo3p-GFP and 5-FOA sensitive. Cells cotransformed with Glo3p-GFP and Sec21p-RFP or Sec7-RFP show coincidence of the green and red fluorescent signal in the punctate pattern typical of the Golgi of S. cerevisiae. B. Glo3p-GFP was expressed in a diploid glo3Δ/glo3Δ gcs1Δgcs1Δ background. This strain was transformed with a plasmid for over-expression of the Sec26p (residues E594-V973; pRC2629b) or Sec21p appendage (residues L676-Q935; pRC2628a) by a copper-inducible promoter (P_CUP1). Cells were grown to early log phase, the cultures were divided, and treated with or without 0.7 mM CuSO₄ for 3 hours at room temperature prior to microscopy. C. Selected sec26^ts mutant strains (sec26-1, sec26-2, and sec26^FW) were transformed with the glo3 truncation and point mutation constructs under control of P_GAL1/10 and plated on SCG as a dilution series to evaluate the suppressive ability of the constructs. D. GLO3 suppression of sec26^ts (sec26-1 and sec26-2) requires the ISS motifs located in the COOH-terminus of Glo3p. Mutant sec26 strains were transformed with the indicated constructs and tested for suppression as in 5C. The mutant ISS motif glo3 construct contained alanines at positions 388–390 and 420–422.