Structural Requirements for Function of Yeast GGAs in Vacuolar Protein Sorting, α-Factor Maturation, and Interactions with Clathrin

The gga1Δ gga2Δ mutant is defective for α-factor maturation and shows a decrease in Kex2p levels. (A) Secretion of mature α-factor in thegga1Δ gga2Δ mutant. Wild-type (MATα), gga1Δ gga2Δ (MATα) mutant, and wild-type (MATa) (negative control) cells were serially diluted to concentrations indicated, and equal amounts were spotted on rich medium (YEPD) containing a freshly spread lawn of α-factor-supersensitive mutant strain RC634 (MATa sst1-3). The agar plate was then incubated at 30°C to assess α-factor secretion of test strains as indicated by relative growth inhibition of sst1-3 mutant cells (halo). (B) Processing of α-factor in the gga1Δ gga2Δ mutant. Strains described for panel A were pulse-labeled at 25°C for 7.5 min as described in Materials and Methods. Various α-factor Golgi-processing intermediates and mature α-factor were immunoprecipitated with anti-α-factor antibodies, resolved by SDS-PAGE (4 to 20% acrylamide gels), and analyzed by fluorography. pαf (pro-α-factor) denotes a range of high-molecular-mass α-factor intermediates (∼90 to 150 kDa) containing the pro-region and Golgi complex-derived, complex glycosylation. unglyc and core refer to unglycosylated (∼20-kDa) and ER-localized, core-glycosylated (∼26-kDa) forms of pro-α-factor, respectively. inter-αf and mαf represent a range of low-molecular-mass α-factor intermediates (probably the α-factor tetrapeptide in various states of processing but with the prosequence removed) (∼6 to 8 kDa) and the 13-aa terminally processed (mature-secreted) form (∼2 kDa), respectively. Relative positions of molecular mass markers are indicated on the right. (C) Steady-state levels of Kex2p in the gga1Δgga2Δ mutant. Whole-cell lysates were prepared from wild-type, gga1Δ gga2Δ, and vps1cells expressing epitope-tagged Kex2p. Proteins were resolved by SDS-PAGE (8% acrylamide gels) and analyzed by immunoblotting using anti-HA and anti-Kar2p (to assess relative protein loading) antibodies as described in Materials and Methods. Positions of Kex2p and Kar2p and molecular mass size markers are indicated.

GGA domains are differentially required for vacuolar sorting. (A) Schematic representation of GGA1 and GGA2 mutants analyzed. Plasmids encoding wild-type Gga2p, Gga2p mutants containing truncations of one or more designated domains and GAT point mutant, wild-type Gga1p, and Gga1p GAT point mutant were generated (Table 2 has plasmid descriptions) and transformed into gga1Δgga2Δ mutant cells. The scale indicates the ability of encoded proteins to complement the gga1Δgga2Δ CPY missorting phenotype as determined by relative percentages of CPY detected (i.e., missorted) in the CPY colony blot below (values are listed in Table 3). Range employed: +++, ≤5% (i.e., similar to wild type); ++, 6 to 25%; +, 26 to 50%; +/−, 51 to 90%; −, 91 to 100% (i.e., similar to gga1Δgga2Δ mutant). Asterisks represent N215A and N219A mutations for GGA1 and GGA2, respectively. (B) Complementation ofgga1Δ gga2Δ CPY missorting by GGA2 truncations. Wild-type cells transformed with vector alone andgga1Δ gga2Δ mutant cells transformed with vector alone and plasmids expressing wild-type Gga2p and Gga2p mutants indicated were grown on SC-URA (selective) medium and blotted with a nitrocellulose membrane, followed by immunoblotting (IB) of the membrane with anti-CPY antibodies as described in Materials and Methods. (C) Complementation of gga1Δgga2Δ CPY processing defect by GGA2 truncations. Wild-type cells transformed with vector alone (lanes 1 and 2) andgga1Δ gga2Δ mutant cells transformed with vector alone (lanes 3 and 4) and plasmids expressing wild-type Gga2p (lanes 5 and 6) and Gga2p mutants indicated (lanes 7 to 14) were pulse-labeled (P) for 10 min, and labeled proteins were chased for 15 min as described in Materials and Methods. Lysates were subjected to immunoprecipitation with anti-CPY antibodies. Immunoprecipitated proteins were resolved by SDS-PAGE, and forms of CPY are indicated as in Fig. 1A. (D) Complementation of gga1Δgga2Δ CPY missorting by GGA1 and GGA2 GAT point mutants. Wild-type cells transformed with vector alone andgga1Δ gga2Δ cells transformed with vector alone and plasmids expressing wild-type GGA1 and GGA2 and GAT point mutants were examined for CPY mislocalization as described for panel B. (E) Test for possible dominant negative effect forgga2-ΔVHS/GAT mutant protein. Wild-type cells transformed with vector alone and plasmids expressinggga2-ΔVHS/GAT- andgga2-ΔVHS/GAT-containing point mutations in a putative clathrin-binding motif (CBS#1) (Table 2 and Materials and Methods) andgga1Δ gga2Δ mutant cells transformed with vector alone were analyzed as described for panel B for CPY mislocalization. wt, wild type.

GGA domains are differentially required for α-factor maturation. (A) Complementation of gga1Δgga2Δ α-factor secretion defect by GGA2 truncations. Strains described for Fig. 3 were spotted on a lawn of tester strain RC634 (MATa sst1-3). Relative growth inhibition of tester lawn is indicative of level of mature α-factor secretion. (B) Complementation of gga1Δ gga2Δ α-factor-processing defect by GGA2 truncations. Strains described for panel A were pulse-labeled, and processed and mature forms of α-factor were immunoprecipitated using anti-α-factor antibodies. Immunoprecipitated proteins were resolved by SDS-PAGE and analyzed, and forms of α-factor and molecular mass standards are indicated as in Fig. 2B.

Gga1p and Gga2p interact with clathrin in vitro. (A) Alignment of Gga1p and Gga2p hinge domains. Amino acid sequences representing hinge domains of Gga1p and Gga2p are aligned, and positions of putative Gga1p CBSs (CBS#1, LIDFD, and CBS#2, LLDFD) and a putative Gga2p CBS (CBS#1, LIDFN) are indicated. Black and gray shading represents amino acid identity and similarity, respectively. (B) Schematic representation of GST-GGA domain fusions tested for clathrin binding. GST-Gga1p and GST-Gga2p fusions (Table 2 shows details of GST expression constructs) are presented relative to their percent clathrin binding in vitro (as determined from panel C and calculated as described in Materials and Methods). The asterisk represents alanine substitution mutations in CBS#1 and/or CBS#2 of Gga1p and CBS#2 of Gga2p (Materials and Methods) as noted in names of fusions to the left. (C) Assay for in vitro clathrin binding. GST alone, GST fusions presented in panel B, and irrelevant GST fusions (as negative controls) were expressed in bacteria, isolated, and incubated with a yeast cytosolic extract, and fusion-bound protein complexes were recovered as described in Materials and Methods. Pull-down samples and trichloroacetic acid precipitants of input lysates and post-pull-down extracts were resolved by SDS-PAGE (8% acrylamide gels) and analyzed by immunoblotting (IB) using anti-Chc1p antibodies (Materials and Methods). Aliquots of GST and GST fusions were resolved by SDS-PAGE and visualized by Coomassie blue staining as a control for levels of fusions used in pull-down experiments.

Functional analysis of GGA hinge, CBS motifs, and GAE domains in vivo. (A) Schematic representation of GGA mutants analyzed. Plasmids encoding wild-type Gga1p and Gga2p and Gga1p and Gga2p mutants containing mutations in their CBS motifs and/or domain truncations were generated (Table 2 shows plasmid descriptions). The relative amount of CPY missorting in gga1Δ gga2Δ transformants (as assessed in panels B and C) is indicated using the scale described for Fig. 3 and based on values in Table 3. The asterisks represent site-directed mutations in CBS#1 and CBS#2 of Gga1p and CBS#2 of Gga2p as noted in names of fusions to the left. Numbers in brackets indicate residues of hinge domains truncated from the respective mutants. (B) Complementation of gga1Δgga2Δ CPY missorting and α-factor-secretion defects by GGA1 mutants. Indicated strains were examined for relative levels of CPY missorting and secretion of mature α-factor as described for Fig.3. (C) Complementation of gga1Δ gga2Δ CPY missorting and α-factor-secretion defects by GGA2 mutants. Indicated strains were examined as described for panel B. WT and wt, wild type.