Grh1 and Bug1 colocalize on the cis-Golgi and form a heterooligomeric complex. (A) Fluorescence micrographs of Grh1-RFP expressed on a CEN plasmid from its own promoter in a Δgrh1 strain with the genomic copy of BUG1 tagged at the N terminus with GFP (RBY85). Representative structures containing both Bug1 and Grh1 are indicated by arrows. (B) Fluorescence micrographs of GFP-Bug1 in strains with or without GRH1 and of Grh1-GFP in strains without or without BUG1. (C) Anti-HA immunoprecipitates from a protease-deficient strain with or without (wt) BUG1 tagged with HA2 in the genome and then probed with the indicated antibodies. (D) Anti-Flag immunoprecipitates prepared from cells with a HA3 tag at the C terminus of genomic BUG1 and containing CEN plasmids without (wt) or with Flag3-Bug1 expressed as in Fig. 1 C. Samples were blotted with the indicated antibodies. (E) Anti-HA immunoprecipitates prepared from a diploid strain without (BY4743; wt) or with (RBY84) one copy of GRH1 tagged C terminally with HA3 and probed for Grh1.

Grh1 binds to the Sec23/24 complex and to reconstituted COPII-coated vesicles. (A) Coomassie-stained gel of anti-Flag precipitates from 1 g of yeast lacking MAK3 or the corresponding wild-type strain, both expressing Grh1-Flag3 from a CEN plasmid as in Fig. 1 C. The indicated bands were identified by mass spectrometry. The bottom Sec24 band presumably corresponds to a degradation product, and Sfb2 was in a mixture with the heat-shock protein Ssb1. (B) 10% of the samples used in A were blotted with an anti-Sec23 antiserum. (C) Reconstituted COPII budding assays with indicated membranes and purified COPII proteins. One tenth of a total reaction (T) and budded vesicles produced in the absence (−) or presence (+) of COPII proteins were immunoblotted for the indicated ER and vesicle marker proteins. Grh1 (arrowhead) migrated just below a major cross-reactive species that was present in all three strains. Grh1 (arrow) migrated just ahead of a prominent background band. WT, wild type. (D) Level of [³⁵S]gpαf packaged into freely diffusible COPII vesicles in an in vitro budding reaction. The reaction was performed with no addition (white bars), with the addition of purified COPII proteins (gray bars), or the same with the tethering factor Uso1 (black bars). The latter tethers COPII vesicles to the Golgi and reduces the diffusible pool. (E) Overall ER/Golgi transport of [³⁵S]gpαf with washed membranes and no addition (white bars) or with the addition of purified factors (COPII, Uso1, and LMA1) to reconstitute transport (gray bars). Error bars represent SD.

The N-terminal amphipathic helix of Grh1 is conserved in other fungal homologues. (A) Alignment of the N termini of S. cerevisiae Grh1 and its relatives in Saccharomyces castellii, Candida glabrata, Ashbya gossypii, Kluyveromyces waltii, Kluyveromyces lactis, Aspergillus fumigatus, Gibberella zeae, Debaryomyces hansenii, Yarrowia lipolytica, and Schizosaccharomyces pombe. The N termini of Mus musculus GRASP65 and GRASP55, Xenopus laevis GRASP55, and the single orthologues in Caenorhabditis elegans (Y42H9AR.1) and Drosophila melanogaster (CG7809) are also shown. The second PDZ-like repeat from M. musculus GRASP55 (G55B) is meant to indicate where the first repeat starts. The hydrophobic residues that form an amphipathic helix are conserved (dots). Residues are shaded where half or more are identical (black) or similar (gray). (B) Helical wheel representation of the N terminus of Grh1 and the hydrophobic (yellow) and hydrophilic (purple) amino acids. (C) Fluorescence micrographs of live yeast expressing RFP-Rud3 from a constitutive form of the PHO5 promoter on a CEN plasmid (Gillingham et al., 2004). Cells are BY4741 with genomic GRH1 tagged with C-terminal GFP, and these and all other images in the paper were obtained on a wide-field microscope as described in Materials and methods. Representative structures containing both Rud3 and Grh1 are indicated by arrows. (D) Fluorescence micrographs of Grh1-GFP and GFP-Imh1 in live cells lacking a genomic copy of MAK3 or SYS1 and in the corresponding wild-type (wt) BY4741 strain. Imh1 targeting is dependent on Arl3, which binds to Sys1 in a Mak3-dependent manner (Behnia et al., 2004; Setty et al., 2004). (E) Fluorescence micrographs of BY4741 cells (wt) or the same lacking MAK3, expressing RFP-Rud3 as in C. (F) Fluorescence micrographs of BY4741 cells lacking GRH1 and expressing wild-type or F2A Grh1-GFP from CEN plasmids as in C.

The binding site for Grh1 is located at the C terminus of Bug1. (A) Alignment of Bug1 with its homologues in other yeasts. The locations of the truncations are indicated. Residues are shaded where half or more are identical (black) or similar (gray). (B) Coiled-coil prediction for Bug1 (MacStripe) along with Bug1 truncations that were tagged at the N terminus with HA3 and expressed as in Fig. 1 C in a Δbug1 strain (RBY46) to examine the effect on the localization of Grh1-GFP. (C) Anti-HA immunoprecipitates from a bug1Δ strain expressing HA3-tagged Bug1 truncations as in B. Lysates and precipitates were probed with anti-Grh1 or -HA antibodies.

Grh1, Bug1, and Mak3 are required to allow suppression of loss of the early Golgi transport components Uso1 or Ypt1. (A) Serial dilutions of uso1Δ/pUSO1 pSLY1-20, uso1Δbug1Δ/pUSO1 pSLY1-20, or uso1Δgrh1Δ/pUSO1 pSLY1-20 strains spotted on −URA −LEU or 5-fluorootic acid (5-FOA) plates and incubated at 30°C. 5-fluorootic acid is toxic to cells expressing Ura3. (B) uso1Δ/pUSO1 pSLY1-20 and uso1Δmak3Δ/pUSO1 pSLY1-20 were streaked on −URA −LEU or 5-fluorootic acid plates and incubated at 30°C. (C) ypt1Δ/pYPT1 pSLY1-20, ypt1Δbug1Δ/pYPT1 pSLY1-20, or ypt1Δgrh1Δ/pYPT1 pSLY1-20 strains were streaked on −URA −LEU or 5-fluorootic acid plates and incubated at 25°C because SLY1-20 does not rescue the ypt1Δ phenotype at 30°C.