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. 2005 Feb;16(2):532-49.
doi: 10.1091/mbc.e04-07-0549. Epub 2004 Nov 24.

The yeast par-1 homologs kin1 and kin2 show genetic and physical interactions with components of the exocytic machinery

Maya Elbert  1 , Guendalina Rossi, Patrick Brennwald
Affiliations

The yeast par-1 homologs kin1 and kin2 show genetic and physical interactions with components of the exocytic machinery

Maya Elbert et al. Mol Biol Cell. 2005 Feb.

Abstract

Kin1 and Kin2 are Saccharomyces cerevisiae counterparts of Par-1, the Caenorhabditis elegans kinase essential for the establishment of polarity in the one cell embryo. Here, we present evidence for a novel link between Kin1, Kin2, and the secretory machinery of the budding yeast. We isolated KIN1 and KIN2 as suppressors of a mutant form of Rho3, a Rho-GTPase acting in polarized trafficking. Genetic analysis suggests that KIN1 and KIN2 act downstream of the Rab-GTPase Sec4, its exchange factor Sec2, and several components of the vesicle tethering complex, the Exocyst. We show that Kin1 and Kin2 physically interact with the t-SNARE Sec9 and the Lgl homologue Sro7, proteins acting at the final stage of exocytosis. Structural analysis of Kin2 reveals that its catalytic activity is essential for its function in the secretory pathway and implicates the conserved 42-amino acid tail at the carboxy terminal of the kinase in autoinhibition. Finally, we find that Kin1 and Kin2 induce phosphorylation of t-SNARE Sec9 in vivo and stimulate its release from the plasma membrane. In summary, we report the finding that yeast Par-1 counterparts are associated with and regulate the function of the exocytic apparatus via phosphorylation of Sec9.

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Figures

Figure 1.
Figure 1.
Isolation of KIN1 as a suppressor of the RHO3 deletion (rho3Δ). (A) Scheme shows genomic DNA fragments identified as suppressors of the rho3Δ phenotype. Cells containing a single copy of the galactose-inducible RHO3 were transformed with a genomic library on a high copy vector and cultured in the presence of 2% glucose to repress Rho3 expression. Plasmids from transformants exhibiting normal growth in the absence of RHO3 were isolated and sequenced. The open reading frame of KIN1 alone was shown to be sufficient for the full suppression of rho3Δ. (B) KIN1 suppresses the cold sensitivity of the rho3-V51 mutant. The rho3-V51 mutant was transformed with KIN1 on a multicopy plasmid, cultured on selective media at 25°C, replica plated from the microtiter plates to YPD media, and incubated at permissive (25°C) and restrictive (14°C) temperatures.
Figure 2.
Figure 2.
KIN1 and KIN2 suppress late secretory mutants. KIN1 and KIN2 suppress the cold sensitivity of the sec4-P48 mutant (A) and the temperature sensitivity of the sec15-1 mutant (B). Mutants were transformed with KIN1 and KIN2 on high copy plasmids, cultured on selective media at 25°C, replica plated from the microtiter plates to YPD media, and incubated at restrictive (14°C for sec4-P48 and 35°C for sec15-1) and permissive (25°C) temperatures.
Figure 3.
Figure 3.
Analysis of the structural requirements for suppression of the secretory mutants by KIN2. (A) Schematic representation of the structure of Kin2 (conserved regions are indicated in red) and Kin2 mutants. Five constructs were examined. KIN2, wild-type; KIN2-NT, a mutant lacking the C-terminal domain; kin2-CT, a mutant lacking the kinase domain; kin2-KD, a kinase-dead mutant with a lysine to methionine substitution at residue 128 (the asterisk indicates the position of the point mutation); and KIN2-Δ42, a mutant with a deletion of the conserved 42-amino acid C-terminal tail. (B) Suppression assays show that the catalytic activity of Kin2 is essential for its function and that the conserved 42-amino acid tail plays a role in autoinhibition. Constructs described above along with the high copy vector control were transformed into sec4-P48, sec15-1, sec1-1, sec2-41, and sec10-2 mutant strains, plated on YP-D media and cultured at permissive (25°C) and their respective restrictive temperatures. kin2-KD and kin2-CT failed to suppress the growth defect of all sec mutants tested. KIN2-Δ42 and KIN2-NT exhibited suppression of sec1-1 at 35°C and sec2-41 and sec10-2 at 37°C, whereas KIN2 suppresses these mutants at 33°C only. (C) In vitro binding of Kin2 C terminus to the N-terminal Kin2 kinase domain demonstrates an intramolecular interaction that depends on the 42-amino acid tail. Kin2-CT or Kin2-CTΔ42 proteins (as shown in A) were in vitro translated in reticulocyte lysates and bound to GST fusion proteins immobilized on glutathione agarose beads. After binding, beads were washed, and the bound fraction was boiled in sample buffer and analyzed by SDS-PAGE and autoradiography. Input lane represents 10% of the radiolabeled protein. (D) Diagram depicting the proposed autoinhibitory intramolecular interaction between the N- and C-terminal domains of Par-1 family kinases.
Figure 4.
Figure 4.
The catalytic domain of SNF1, but not of other kinases belonging to the Snf1 family, suppresses the same set of late secretory mutants as KIN1 and KIN2. The sec15-1, sec10-2, and sec2-41 mutants were transformed with either vector alone or the respective catalytic domains of KIN2 (encoding residues 1-526), SNF1 (1-432), HSLl1 (1-462), GIN4 (1-432), KCC4 (1-437), YPL141c (1-387), KIN4 (1-366), and YPL150w (1-426) on high copy plasmids. Growth of transformants at respective restrictive temperatures was tested.
Figure 5.
Figure 5.
Kin1 and Kin2 proteins are found in both cytosolic and membrane-bound pools. (A) Affinity-purified antibodies against the C-terminal domains of Kin1 and Kin2 recognize proteins in a specific manner, running at ∼145 kDa on SDS-PAGE. Wild-type cells and cells containing either KIN1 or KIN2 on high copy were subjected to 7% SDS-PAGE and Western blot by using affinity-purified antibodies against Kin1 and Kin2. (B) Kin1 and Kin2 are found in both the cytosolic and membrane-bound pools. Lysates from cells containing either vector alone, KIN1, or KIN2 on high copy were treated with or without Triton X-100 and centrifuged at 30,000 × g for 15 min. Supernatant and pellet fractions were analyzed (T, total; S30, supernatant; P30, pellet) by SDS-PAGE (pellet fractions were resuspended in the volume equal to that of S30 fraction) and blotted with anti-Kin1 and anti-Kin2 antibodies. Approximately 70% of both Kin1 and Kin2 is seen in the cytosolic fraction and ∼30% is in a Triton-sensitive membrane fraction. Samples also were immunoblotted with anti-Sso1/2 polyclonal antibody as an internal control of the fractionation procedure (consistent with previous reports ∼80% of Sso1/2 was detected in the pellet fraction).
Figure 6.
Figure 6.
Kin1 and Kin2 physically associate with the t-SNARE Sec9 and the Sec9-binding protein Sro7. (A) Antibodies against Kin1 and Kin2 coimmunoprecipitate Sec9. Cell lysates carrying either high copy KIN1 and SEC9 or high copy KIN2 and SEC9 were subjected to native immunoprecipitation with preimmune serum (PI) and either anti-Kin1 or anti-Kin2 antibodies (I), respectively, as described in Materials and Methods. Samples were separated on 8% SDS-PAGE and immunoblotted with anti-Sec9 and either anti-Kin1 or anti-Kin2 antibodies, respectively. “T” stands for total protein in the lysate before IP, loaded at 1:20 to the amount of lysate used to immunoprecipitate Kin1 and Kin2 in PI and I lanes. (B) Cross-linking experiment confirms association between Kin1 and Sec9. Cells expressing SEC9 and KIN1 on a multicopy plasmid were 35S-labeled for 1 h and lysed osmotically in PBS. Lysates were subjected to cross-linking, followed by two rounds of immunoprecipitations, initially with α-Kin1 and α-Sec9 antibodies, followed by a denaturing IP with α-Kin1 and α-Sec9 antibodies. Samples were run on 7% SDS-PAGE, and the 35S-labeled protein was detected by autoradiography. (C) Kin2 coassociates with Sro7, but not with Snc1, Sso2, or Sec4. Lysates from cells transformed with high copy KIN2 were subjected to native immunoprecipitation with preimmune serum (PI) and α-Kin2 antibody (I) as described in Materials and Methods, followed by SDS-PAGE and immunoblot with α-Kin2, α-Sro7, α-Snc1, α-Sso2, and α-Sec4 antibodies.
Figure 7.
Figure 7.
The t-SNARE Sec9 is phosphorylated as an effect of Kin1 or Kin2 induction. (A) Sec9 undergoes a phosphorylation-dependent size shift in response to Kin2 induction. Lysates from galactose-induced yeast cells containing high copy Sec9 and either a CEN/GAL vector or CEN/GAL-KIN2 were immunoprecipitated with anti-Sec9 antibody. Immune complexes were treated with λ-phosphatase and CIP for 30 min at 30 and 37°C, respectively. Untreated and mock-treated samples, incubated under identical conditions, were used as a control. Samples were subjected to 8% SDS-PAGE and immunoblotted with α-Sec9 antibody. (B) N terminus of Sec9 is a substrate of Kin2 in vitro. Cells expressing Kin2 were subjected to immunoprecipitation with either α-Kin2 antibody (I) or preimmune serum (PI) as a negative control. Immobilized Kin2 on protein A-Sepharose beads was used for in vitro kinase assays. Equal molar amounts (1.5 μM) of soluble recombinant Sec9-NT1 (amino acids 1-168), Sec9-NT2 (amino acids 166-401), and Sec9-CT (amino acids 402-651) were added to the reaction and incubated with [32P]ATP for 30 min at 30°C. Samples were analyzed by SDS-PAGE and autoradiography (1). In 2, wild-type Sec9-NT2 and Sec9-NT2-S315A mutant were tested in an in vitro kinase reaction as described above. Concentrations of recombinant proteins used in the kinase reaction were measured by a Bradford protein assay, normalized to equal concentrations, and verified by Coomassie staining. (C) Sec9 is not a direct target of Kin2 in vivo. Lysates from cells containing CEN/GAL vector or CEN/GAL Kin2 containing multicopy wild-type Sec9 or mutant Sec9-S315A were induced with galactose for 4 h. Cells were then spheroplasted and lysed, and the lysates were boiled and separated by SDS-PAGE and immunoblotted with anti-Sec9. Arrows indicate Sec9 mobility shift.
Figure 8.
Figure 8.
Sec9 is the only component of the exocytic apparatus tested that is phosphorylated upon Kin2 induction. Cells containing empty vector, or GAL-KIN2 were induced with galactose for 2 h before labeling with either [35S]methionine or [32P]orthophosphate. Denatured cell lysates were prepared and immunoprecipitated with antibodies to the components of the late secretory machinery. Phosphate-incorporation was detected by SDS-PAGE and autoradiography. Quantitation demonstrated that the when cells were induced with GAL-KIN2, the amount of 32P label incorporated into Sec9 increased threefold. The small increase in 32P labeling in the cells lacking GAL-KIN2 was due to slight increase in overall 32P labeling in this sample.
Figure 9.
Figure 9.
Kin1 overexpression reduces the membrane association of Sec9 while increasing secretory function. (A) Fractionation of Sec9 in GAL-KIN1 induced cells. Lysates from galactose-induced and uninduced cells containing CEN/GAL KIN1 and high copy SEC9 were treated with or without Triton X-100 and centrifuged at 30,000 × g for 15 min. Supernatant and pellet fractions were subjected to SDS-PAGE and blotted with anti-Sec9 antibodies (T, total; S30, 30k × g supernatant; P30, 30k × g pellet). The proportion of Sec9 in the cytosolic and Triton-sensitive membrane fraction was quantified as an average of three independent experiments. Samples also were immunoblotted with anti-Sso1/2 polyclonal antibody as an internal control. (B) Suppression of the sec1-1 growth defect by GAL-KIN1. sec1-1 mutants were transformed with empty vector or GAL-KIN1 and replicated for growth at permissive (25°C) and restrictive (33°C) temperatures on media that induces (YP-Gal) or represses (YPD) overexpression of the Kin1 protein. (C) Suppression of the sec1-1 secretion defect by GAL-KIN1. sec1-1 mutant strains containing empty vector or GAL-KIN2 were induced for 2 h in galactose, shifted to 33°C for 2 h, and then processed for BglII secretion (defects in secretion are seen as an increase in the fraction of BglII found internally).
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