The Arrest of Secretion Response in Yeast Jayasri Nanduri, Alan M Tartakoff  Molecular Cell  Volume 8, Issue 2, Pages 281-289 (August 2001) DOI: 10.1016/S1097-2765(01)00312-4

Figure 1 Localization of a Nucleolar Protein and Nucleoporins sec1-1 (ATY1217) was studied at 23°C or after a shift for 2 hr to 37°C ± cycloheximide (100 μg/ml). The nucleolar protein Fpr3p and the nucleoporin Nsp1p were localized by indirect immunofluorescence. Note the major relocation of each protein. Relocation is inhibited when cycloheximide is included. Each of the proteins returns to its normal location upon reincubation at 23°C (data not shown). The bottom panels document the constancy of Fpr3p during relocation by Western blotting. (Note: the magnification of the images in this figure is greater than in the following figures.) Molecular Cell 2001 8, 281-289DOI: (10.1016/S1097-2765(01)00312-4)

Figure 2 Localization of Fpr3p in ts-Strains which Affect Membrane Traffic Each strain (vps1-100ts [SNY4], vps18-1ts [SRY18T-1], vps45ts-3 [RPY15], and end4ts [LCY16]) was grown in YEPD at 23°C and studied after incubation for 2 hr at 37°C. The vps mutants are ts for delivery of proteins to the vacuole, and end4 is ts for endocytosis. Nuclear proteins do not relocate in these strains Molecular Cell 2001 8, 281-289DOI: (10.1016/S1097-2765(01)00312-4)

Figure 3 Involvement of Pkc1p in the ASR (A) Relocation of Fpr3p requires Pkc1p. Relocation of the nucleolar protein, Fpr3p, occurs in sec1-1 (ATY1217) but not in sec1-1 pkc1-Δ (ATY1381) upon incubation for 2 hr at 37°C. Note: in order to study strains which lack normal Pkc1p activity, cells must be grown with osmotic support (0.5 M sorbitol). Since the relocation of nuclear proteins in sec1-1 Pkc1+ cells is not affected by growth in this medium, it is unlikely that it complicates the analysis. (B) A Pkc1p active site mutant does not signal. sec1-1 pkc1-Δ (ATY1381) carrying plasmids encoding wild-type Pkc1p-HA or an active site mutant, Pkc1pK853R-HA, were studied at 23°C or after shift to 37°C for 2 hr. The panels at the top show that Fpr3p does not relocate unless Pkc1p is active. For these panels, since expression is driven by a galactose-inducible promoter, cells were pregrown in raffinose dropout medium containing 0.5 M sorbitol at 23°C, 2% galactose was added for 3 hr and cells were then shifted to 37°C for 2 hr. Localization of tagged forms of Pkc1p (middle panels). Note that wild-type Pkc1p-HA is broadly distributed at 23°C but the inactive site mutant, Pkc1pK853R-HA, concentrates at sites of bud formation (stars). After the shift to 37°C for 2 hr, wild-type Pkc1p is excluded from the nucleus (arrows), while Pkc1pK853R-HA is not. This is expected since signaling does not occur in the absence of Pkc1p activity. Nuclei were colocalized by staining with DAPI as shown in the bottom panels. (C) A Pkc1p putative DAG binding site mutant does not signal. The panels at the top show that the DAG binding site mutant (4C/S) does not mediate relocation of Fpr3p. For these panels, in which the normal PKC1 promoter was present, cells were grown in glucose dropout medium at 23°C supplemented with 0.5 M sorbitol. Western blotting with the anti-HA antibody of the same samples illustrated in Figure 3C shows that Pkc1p4C/S-HA is as abundant as Pkc1p-HA under the conditions studied Molecular Cell 2001 8, 281-289DOI: (10.1016/S1097-2765(01)00312-4)

Figure 4 Role of the Pkc MAP Kinase Cascade in the ASR (A) Western blot. Cell lysates were probed with the phosphospecific p44/42 MAP kinase Ab (NEBiolabs #9101) to evaluate phosphorylation of the terminal kinase of the PKC path, Mpk1p/ Slt2p (Verna et al., 1997). wild-type (YPH500) and sec1-1 (ATY1217) were grown in YEPD and incubated at 37°C for the indicated times. Note that both wild-type and sec1-1 show a similar modest increase of Mpk1p phosphorylation after 30 or 60 min at 37°C. (B) Relocation of Fpr3p does not require the Pkc MAP kinase cascade. Relocation of the nucleolar protein Fpr3p is seen in both sec1-1 (ATY1217) and sec1-1 mpk1-Δ (ATY1380) over 2 hr at 37°C. (C) Conditions known to stimulate the Pkc MAP kinase cascade do not relocate Fpr3p. Localization of Fpr3p in wild-type (YPH500) subjected to 10 min hypotonic shock at 23°C (addition of 4 volumes of water) or to heat shock (37°C for 2 hr). Cells were grown at 23°C in YEPD Molecular Cell 2001 8, 281-289DOI: (10.1016/S1097-2765(01)00312-4)

Figure 5 Studies of Wsc2p (A) Relocation requires Wsc2p. Relocation of Fpr3p occurs in sec1-1 (ATY1217) but not in sec1-1 wsc2-Δ (ATY1383) when incubated for 2 hr at 37°C. Cells were pregrown in YEPD at 23°C. (B) Wsc2p along the Secretory Path Is Required for the ASR Localization of Nsp1p (panels 1–4), Fpr3p (panels 1′–4′) and Wsc2p-myc (panels 1″–4″) in sec1-1 wsc2-Δ [pGAL-WSC2-myc] (ATY1383) in four different situations. In each case, cells were grown in raffinose dropout medium at 23°C. Panels 1, 1′, 1″: cells after addition of 2% galactose for 2 hr at 23°C, which allows most Wsc2p to reach the sites of bud formation (arrows). Panels 2, 2′, 2″: cells were preinduced for 2 hr at 23°C to position Wsc2p at the cell surface, and then shifted to 37°C upon addition of glucose for 2 hr (to stop Wsc2p synthesis). Panels 3, 3′, 3″: cells after shift to 37°C for 2 hr without induction of Wsc2p synthesis. Panels 4, 4′, 4″: cells after addition of 2% galactose for 2 hr at 37°C, to trap Wsc2p in secretory vesicles, which are visualized as a “haze.” Note that Nsp1p and Fpr3p relocate only when Wsc2p is trapped in secretory vesicles (panels 4, 4′). (C) Western blot. Levels of Wsc2p-myc under conditions (panels 1–4) illustrated in Figure 5B Molecular Cell 2001 8, 281-289DOI: (10.1016/S1097-2765(01)00312-4)

Figure 6 Model of Signaling Path The designation “Wsc” refers to Wsc1p and Wsc2p along the secretory path. The MAP kinase cascade—as explained in the text—is not required for the ASR Molecular Cell 2001 8, 281-289DOI: (10.1016/S1097-2765(01)00312-4)