KIN1 and KIN2 Are Potent Suppressors of Secretion-defective Alleles of Rho3 and Cdc42
Rho3 is a member of the Rho family of GTPases that exhibits multiple genetic and physical interactions with components of the exocytic machinery and is itself required for efficient exocytosis (Adamo et al., 1999
; Robinson et al., 1999
). To isolate candidate downstream effectors of RHO3
function in exocytosis, we performed a genetic screen for dosage suppressors of the slow growth phenotype of a RHO3
deletion strain (rho3
Δ). We constructed a strain containing the RHO3
coding sequence under the control of the glucose-repressible GAL
promoter as the only source of Rho3 in the cell. This strain, which grows normally on galactose-containing media but extremely slowly in glucose-containing media, was transformed with a yeast genomic library prepared in a multicopy vector, and transformants were selected for growth on glucose-containing medium. Plasmids from colonies growing on glucose were isolated, retested for suppression, and then analyzed by sequencing. From this analysis, we identified 25 suppressing plasmids containing overlapping parts of six distinct loci. Five of the six loci had genes previously isolated as dosage suppressors of a rho3
Δ mutant: BEM1
(1 isolate), SRO9
(1 isolate), SEC4
(2 isolates), SSO2
(3 isolates), and RHO3
itself (17 isolates) (Imai et al., 1996
; Adamo et al., 1999
). Subcloning of the various regions in the sixth locus identified the suppressing gene to be coincident with the KIN1
gene (). Although several suppressors of rho3
Δ (SEC4, SRO7, SRO77, SEC9
, and SSO2
) are known components of the late secretory machinery, the function of KIN1
is not known. The KIN1
open reading frame alone was sufficient to suppress rho3
Δ, and thus we identified KIN1
as a novel dosage suppressor of Rho3.
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 (more ...)
Mutations in RHO3
affect both actin organization and post-Golgi vesicle transport (Imai et al., 1996
; Adamo et al., 1999
). The rho3-V51
cold-sensitive mutant has a secretory defect in the absence of functional and structural perturbations of the actin cytoskeleton; hence, this mutant affects a function of Rho3 in exocytosis that is independent of actin (Adamo et al., 1999
). To determine whether Kin1 functions specifically in the secretory pathway downstream of Rho3, we assessed the ability of KIN1
to suppress the rho3-V51
is viable at 25°C, but not at 14°C. shows that overexpression of KIN1
resulted in restoration of growth of the rho3-V51
mutant at the restrictive temperature (14°C). In addition, we have previously reported that the growth defect of cdc42-6
, the secretion-impaired mutant of another Rho GTPase, is specifically suppressed by introduction of KIN1
on the multicopy plasmid (Adamo et al., 2001
). This suppression was specific to cdc42-6
, because KIN1
failed to suppress more pleiotropically defective alleles of CDC42
such as cdc42-1
Because the coding sequence of KIN2 is similar to that of KIN1 (51% identity), we asked whether they shared a common function downstream of Rho3 and Cdc42 by determining the ability of high-copy KIN2 to suppress the growth defects associated with rho3-V51 and cdc42-6 mutant strains. As shown in , we found that KIN2 strongly suppressed the growth defect of these mutants at their respective restrictive temperatures (14°C for rho3V-51 and 32°C for cdc42-6), and the potency of KIN2 was identical to that of KIN1. The fact that both kinases specifically suppress secretory-defective alleles of Rho3 and Cdc42 suggests that both KIN1 and KIN2 act in the secretory pathway downstream of the Rho GTPases.
KIN1 and KIN2 act redundantly in suppression of rho3-V51 and cdc42-6, secretion-impaired mutants of Rho GTPases
KIN1 and KIN2 Exhibit Genetic Interactions with Components of the Late Secretory Machinery
The ability of KIN1
to suppress the cdc42-6
alleles suggests a possible role for these kinases in the regulation of exocytosis. To further explore this idea, we examined the ability of multicopy KIN1
to suppress the temperature-sensitive growth defect of a number of secretory (sec
) mutants. An example of the suppression analysis, summarized in , is shown in . Elevated dosage of KIN1
restored the growth of the late sec
to wild-type levels at 14 and 35°C, respectively (). sec4-P48
is a cold-sensitive effector domain mutant of the Rab GTPase SEC4
(Brennwald et al., 1994
), whereas sec15-1
is a temperature-sensitive mutant of one of the components of the Exocyst complex, Sec15 (TerBush and Novick, 1995
). We examined the secretory defect in sec15-1
cells with multicopy KIN2
compared with control sec15-1
cells containing empty vector after a shift to the restrictive temperature. This analysis demonstrated that although unsuppressed sec15-1
cells are found to accumulate 36% of Bgl
II internally, the accumulation is reduced to wild-type levels in sec15-1
cells containing high-copy KIN2
where only 18% of Bgl
II is found internally. Therefore, the suppression of the growth defect was found to correlate closely to suppression of the secretion defect in these cells. Suppression analysis demonstrated that KIN1
also suppress the growth defect of a number of other late secretory mutants. These include the temperature-sensitive mutants of two additional components of the Exocyst complex; Sec3 (sec3-2
) and Sec10 (sec10-2
), which were rescued at the nonpermissive temperature of 35°C by introduction of KIN1
on high copy. Also, we show that expression of multicopy KIN1
restores the growth of sec1-1
at 33°C and sec2-41
at 33°C. sec1-1
is a mutant of SEC1
, which is involved in SNARE assembly (Carr et al., 1999
), and sec2-41
is a mutant in SEC2
, the nucleotide exchange factor for Sec4 (Walch-Solimena et al., 1997
). Thus, KIN1
exhibit strong genetic interactions with multiple components of the exocytic machinery. The suppression profile of KIN1
was identical to that of KIN2
, providing additional evidence in favor of the functional redundancy of the two kinases (). The ability of KIN1
to suppress several late sec
mutant genes indicates that they function downstream of these proteins at the later stage of exocytosis.
Summary of late sec mutants suppression by KIN1 and KIN2
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 (more ...)
Comparison of suppression properties of high copy KIN1, KIN2 with those of high copy SEC9
However, we found that KIN1
do not suppress the temperature-sensitive phenotype of the sec9-4
mutant (), a mutant of the t-SNARE Sec9 that is defective in SNARE complex assembly and that is required for vesicle fusion with the plasma membrane (Rossi et al., 1997
), nor do they suppress the growth defect of sro7/77
Δ, a mutant with a double disruption of genes encoding Sec9-binding proteins Sro7 and Sro77 (). These observations place Kin1 and Kin2 function downstream of polarized vesicle delivery and upstream of the terminal fusion event.
Analysis of the Structural Requirements for KIN1 and KIN2 Function in the Secretory Pathway
To elucidate the nature of Kin1 and Kin2 function in the secretory pathway, we determined the minimal domain requirement that confers suppression. Kin1 and Kin2 contain a kinase domain at the N terminus of the protein and a regulatory domain at the C terminus (). The kinase domain as well as the 42-amino acid stretch on the extreme carboxy terminus are highly conserved between Kin1 and Kin2 and a number of their orthologues from other species. Because Kin1 and Kin2 proteins display structural and functional redundancy, we focused on Kin2. Mutant KIN2
constructs with deletions of the kinase domain or the 42 amino acid C-terminal tail were designed to assess the significance of these domains for Kin2 function in the secretory pathway. The following mutants were generated: KIN2-NT
, lacking the regulatory C-terminal domain of the protein; kin2-CT
, lacking the catalytic N-terminal domain; kin2-KD
, the kinase-dead mutant, where a single critical Lys128
residue in the second catalytic domain (conserved residue mapped by kinase sequences alignment; Hanks et al., 1988
) was mutated to a Met; and finally the KIN2-
mutant, with a deletion of the conserved 42 amino acid C-terminal tail ().
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; (more ...)
Function was assayed by analysis of the suppression properties of the KIN2 mutants expressed at high copy. The suppression of the mutant phenotype of several late sec genes was tested, including sec15-1 and sec4-P48, sec1-1, sec2-41, and sec10-2 (). Wild-type KIN2 on a multicopy plasmid behaved as reported above ( and ), restoring the viability of these mutants at certain restrictive temperatures. The kinase-inactive KIN2 mutants kin2-CT, lacking the entire kinase domain, and kin2-KD, the kinase-dead mutant, failed to suppress the growth defect of all sec mutants tested (). These data demonstrate that the catalytic activity of Kin2 is critical for its function in the secretory pathway.
Multicopy expression of KIN2-NT, lacking the entire C-terminal domain, and KIN2-Δ42, lacking the 42 amino acid C-terminal tail, rescued the growth phenotype of all late sec mutants tested: sec15-1, sec4-P48, sec1-1, sec2-41, and sec10-2 (). Thus, the regulatory domain of Kin2 is functionally dispensable. Furthermore, we observed that the KIN2-NT and KIN2-Δ42 mutants at high copy gain the ability to suppress several secretory mutants: sec1-1, sec2-41, and sec10-2, at temperatures at which the wild-type KIN2 failed to suppress (). The sec1-1 and sec2-41 temperature-sensitive mutants are suppressed at 33°C and the sec10-2 mutant at 35°C in a comparable manner by KIN2, KIN2-NT, and KIN2-Δ42 (our unpublished data). However, KIN2 fails to suppress sec1-1 at 35°C and sec2-41 and sec10-2 at 37°C, whereas the KIN2-NT mutant with the deletion of the regulatory domain is able to rescue these mutants at the more restrictive temperatures. Remarkably, the truncation of the distal 42 amino acids at the C terminus of KIN2, in KIN2-Δ42, is sufficient to phenocopy the gain of function observed by KIN2-NT. Both mutant forms of KIN2 are capable of suppressing sec1-1 at 35°C and sec2-41 and sec10-2 at 37°C (). These data demonstrate that the C-terminal nonkinase domain of Kin2 acts as a negative regulator of Kin2 function in the secretory pathway and that the conserved 42-amino acid tail is essential for this negative regulatory function.
The greater potency of the Kin2 constructs lacking the distal C-terminal sequence might reflect the acquisition of the catalytically “active” protein conformation in the absence of the putatively inhibitory C-terminal tail. Possibly, in a dormant state the wild-type kinase exists in a closed conformation, with the tail bound to the catalytic core, hindering its activity, until the “ON” regulatory event relieves this autoinhibition (). This hypothesis presupposes the presence of a direct physical interaction between the tail of Kin2 and its kinase domain. To test whether this intramolecular interaction takes place, we used a yeast two-hybrid analysis. We created the following constructs: Kin2-CT and Kin2-CTΔ42 (encoding the regulatory domain with and without the C-terminal tail region, respectively) as GAL4 binding domain fusions and Kin2 (full-length protein), Kin2-NT (kinase domain), Kin2-CT (regulatory domain), Kin2-CTΔ42 (regulatory domain with the deletion of the C-terminal 42 amino acids) and Kin2-Δ42 (full-length protein with the deletion of C-terminal 42 amino acids) as GAL4 activation domain fusions. All constructs in activation and binding domain fusions were expressed at comparable levels as verified by Western blot analysis (our unpublished data). We found that the C-terminal regulatory domain of Kin2 (Kin2-CT in GAL4BD) binds to the catalytic N-terminal domain of Kin2 (Kin2-NT in GAL4AD) (). Moreover, this interaction is mediated by the conserved 42 amino acid tail, because it is abolished by truncation of the tail region in the C-terminal domain of Kin2: Kin2-CTΔ42 does not bind to Kin2-NT. Kin2-CT does not interact with itself, which is consistent with the regulatory domain interacting with the kinase domain only. In addition, although Kin2-CT fails to interact with the full-length Kin2, it shows interaction with the C-terminally truncated Kin2 Kin2-Δ42 (lacking the distal 42 amino acids). This result may signify that Kin2-CT associates with Kin2 in a presumably “open” or active conformation, as in Kin2-Δ42, but not with Kin2 in a closed conformation, as in full-length kinase. Thus, yeast two-hybrid analysis revealed that the regulatory domain of Kin2 binds to its catalytic domain and that the 42-amino acid tail is a prerequisite for this interaction. To further support the possible interaction of the Kin1/2 kinase domain with the C-terminal domain, we examined the ability of the domains to interact in vitro. We made use of a recombinant GST-fusion of the kinase domain and in vitro-translated C-terminal domain fragments containing either the intact C terminus, Kin2-CT (523-1147), or an identical domain lacking the conserved C-terminal 42 amino acids, Kin2-CTΔ42 (523-1106). The results shown in demonstrate that these domains do in fact interact and that this interaction requires the C-teminal 42 residues of the Kin2. Together, the in vitro binding data and the two-hybrid analysis strongly suggest that the C-terminal regulatory domain of Kin2 physically interacts with the N-terminal kinase domain to mediate an autoinhibitory regulation of the kinase. Furthermore, we show that the highly conserved 42-amino acid tail of Kin2 is critical for the both the physical interaction between these domains and for the negative regulatory effect of the C-terminal domain as demonstrated by the effects on suppression of the late sec mutants. Together, this strongly suggests that C-terminal domain of Kin1/2 functions as an autoinhibitory domain and that this autoinhibition requires the highly conserved C-terminal 42 amino acids. This provides the first mechanistic insight into to the function of this highly conserved 42-residue sequence at the C terminus of all Par-1 family kinases.
Yeast two-hybrid analysis shows that C-terminal 42 amino acid tail of Kin2 is necessary for the interaction of the regulatory C-terminal domain of Kin2 with the kinase domain
The Kinase Domain of SNF1, but Not Other Kinases of the CaMK Group, Show Suppression Properties of the Kin1 and Kin2 Kinases
It was previously reported that deletion of either KIN1
is neither lethal nor deleterious for cell growth (Lamb et al., 1991
; Donovan et al., 1994
). Because our data demonstrated functional redundancy of KIN1
, we proceeded to analyze whether the presence of at least one of these genes is required for cell viability. We created strains carrying single or double disruptions of these genes by homologous recombination. Consistent with the previous reports, kin1
Δ and kin2
Δ single disruptant strains were viable. The double disruptant kin1
Δ progeny, obtained by crossing of the kin1
Δ strain with kin2
Δ, demonstrate normal growth at all temperatures tested (25, 14, and 37°C). The single, kin1
Δ and kin2
Δ, and double kin1
Δ disruptants did not exhibit any significant defects in growth or secretion, as confirmed by invertase secretion assays (our unpublished data).
KIN1 and KIN2 orthologues in S. pombe, C. elegans, D. melanogaster, and mammalian cells display pronounced gene disruption phenotypes. Hence, it is likely that in S. cerevisiae anther molecule(s) acts to substitute the compromised function of Kin1 and Kin2. Kin1 and Kin2 belong to the CaMK protein kinase group, members of which share significant sequence similarity in the catalytic domain. Therefore, we examined whether other kinases of this group display functional redundancy with Kin1 and Kin2. BLAST search was used to identify the closest homologues of Kin1 and Kin2, and, as a result, we considered seven proteins of the CaMK group for further analysis, including Snf1, Hsl1, Gin4, Kcc4, Ypl141c, Kin4, and Ypl150w. To determine whether these proteins act in the secretory pathway, we tested their ability to rescue the growth defect of sec2-41, sec10-2, and sec15-1. Because several members of the CaMK group, such as Hsl1, Gin4, and Kcc4 bear a sequence at the distal carboxyl tail almost identical to that of Kin1 and Kin2, we hypothesized that they might be subjected to a similar mode of autoregulation as Kin2. To overcome this possible autoinhibition, we generated deletion constructs of SNF1 (encoding amino acids 1-432), HSL1 (1-462), GIN4 (1-432), KCC4 (1-437), YPL141C (1-387), KIN4 (1-366), and YPL150W (1-426), which lack the regulatory domain while preserving intact all regions important for catalytic activity based on sequence alignment. These constructs were introduced on a multicopy plasmid into sec15-1, sec10-2, and sec2-2 mutants and incubated at permissive and nonpermissive temperatures. We show that the catalytic domain of SNF1 (SNF1-NT) suppresses the growth defect of sec15-1, sec10-2, and sec2-2 mutants (). Furthermore, the suppression of sec mutants by SNF1-NT is comparable to that of KIN2-NT. By contrast, constructs encoding the catalytic domains of Hsl1, Gin4, Kcc4, Ypl141C, Kin4, and Ypl150W failed to restore growth of the sec mutants at restrictive temperatures to any significant degree. Our data demonstrate that Snf1, which is structurally closer to Kin1 and Kin2 than any of the other seven members of the CaMK group, is the only kinase, out of those tested that may function redundantly with Kin1 and Kin2 in exocytosis.
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 (more ...)
To test whether the function of at least one of these genes, KIN1, KIN2
, or SNF1
, is essential for cell viability, we generated the strain with a triple disruption, kin1
Δ was created by substitution of SNF1
with the KanR
(Kanamycin) gene sequence in the context of the diploid strain homozygous for the KIN1
Δ) and heterozygous for the KIN2
Δ). A triple disruptant was obtained via sporulation and tetrad analysis. The triple mutant kin1
Δ as well as the double kin1
Δ, and kin2
Δ mutants displayed slow growth phenotype inherent to the SNF1
deletion alone (Celenza and Carlson, 1984
). The kin1
Δ triple mutant did not display any synthetic defect in growth or secretion (as verified by invertase secretion assays; our unpublished data).
Kin1 and Kin2 Physically Associate with Components of the Late Exocytic Machinery
Because genetic data place Kin1 and Kin2 function to the late secretory pathway, we analyzed whether these proteins physically associate with any of the components of the exocytic machinery.
Initially, we generated antibodies to detect Kin1 and Kin2 and characterized their localization in budding yeast. Antibodies were raised against a region in the C-terminal domain of Kin1 and Kin2. Affinity-purified antisera were tested by Western blot analyses on samples containing either high copy KIN1
or empty vector (). Kin1 and Kin2 antisera specifically recognized each protein and did not cross-react with the other gene product, with both proteins running on SDS-PAGE at ~145 kDa as predicted by the estimated molecular weight (Lamb et al., 1991
; Donovan et al., 1994
). Next, we analyzed the intracellular distribution of Kin1 and Kin2 by cell fractionation. Lysates from cells overexpressing Kin1 or Kin2 were treated with or without Triton X-100 and centrifuged at 30,000 × g
. Supernatant and pellet fractions obtained were analyzed by SDS-PAGE and Western blot with anti-Kin1, anti-Kin2, and anti-Sso1/2 (as an internal control) antibodies (). We determined that ~70% of both Kin1 and Kin2 occur in the supernatant or cytosolic fraction (precisely 71.5% of Kin1 and 73.7% of Kin2 as an average of 3 or more experiments), and the remaining ~30% is found in the pellet or membrane-bound fraction. Therefore, consistent with the previous report (Tibbetts et al., 1994
), Kin1 and Kin2 partition to both cytosolic and membrane-associated pools in S. cerevisiae
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 (more ...)
Genetic data suggest that Kin1 and Kin2 act downstream of Rho3, Cdc42, Sec4, and components of the Exocyst complex but upstream of the t-SNARE Sec9 and the Sec9-binding protein Sro7. Therefore, we hypothesized that Kin1 and Kin2 interact and function together with proteins important for the final stages of exocytosis, such as the SNAREs. To test this, we used the candidate approach to search for Kin1- and Kin2-interacting partners. First, we observed that antibodies against both Kin1 and Kin2 bring down the t-SNARE Sec9 when both genes are expressed at high copy (). Sec9 immunoprecipitation with Kin1 and Kin2 antibodies was detected with the endogenous levels of Kin1 and Kin2 as well (our unpublished data). To confirm that Kin1 exists in a protein complex with Sec9 by a different method, we used a chemical cross-linking procedure. Cells from strains overexpressing both KIN1 and SEC9 were labeled with [35S]methionine for 1 h and then lysed. Lysed cells were treated with the chemical cross-linker DSP and subjected to two rounds of immunoprecipitation. In the first round, samples were divided into two pools and incubated with affinity-purified antibodies either against Kin1 or against Sec9, and immune complexes formed were pulled down via protein A-Sepharose. In the second round, each of the two pools was subjected to a denaturing immunoprecipitation with either anti-Sec9 or anti-Kin1 antibodies. Our results show that in the presence of the cross-linker Kin1 coimmunoprecipitates Sec9, and in its turn, Sec9 pulls down Kin1 ().
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 (more ...)
To identify other potential Kin1- and Kin2-interacting partners, we performed a series of native immunoprecipitation experiments testing for the association of Kin1 and Kin2 with proteins acting in the secretory pathway. Namely, we examined whether antibodies against Kin2 can pull down two other exocytic SNAREs, Sso and Snc, the Sec9-interacting protein Sro7, and the Rab GTPase Sec4. Kin2 was immunoprecipitated from the cell lysates carrying multicopy KIN2, and the sample was analyzed by Western blotting with α-Kin2, α-Sso2, α-Snc1, α-Sro7, and α-Sec4 antibodies, respectively. This experiment revealed that the α-Kin2 antibody brings down significant amounts of Sro7, a homologue of a tumor suppressor protein lethal giant larvae (Lgl), but not other proteins tested (). As expected, Sro7 also coimmunoprecipitates with anti-Kin1 antibodies (our unpublished data). Therefore, Kin1 and Kin2 associate with t-SNARE Sec9 and the Sec9-binding protein Sro7.
To determine whether association of Kin1 and Kin2 with Sec9 and Sro7 is a result of a direct binding between these proteins, we used a yeast two-hybrid analysis. Kin1 and Kin2 in both GAL4 binding and GAL4 activation domain fusions did not show interaction with either Sec9 or Sro7 in GAL4 activation and GAL4 binding fusions, respectively (our unpublished data). This indicates that the interaction of Kin1 and Kin2 with Sec9 and Sro7 is not direct but occurs via other intermediaries in the complex.
The physical association of Kin1 and Kin2 with the SNARE machinery supports the hypothesis that these Par-1 counterparts play a role in exocytosis at a stage between vesicle docking site recognition and fusion with the plasma membrane.
Kin1 and Kin2 Induce Phosphorylation of Sec9 In Vivo and Its Release from the Plasma Membrane to the Cytosol
Next, we focused on finding a downstream target of Kin1 and Kin2 function in the exocytic pathway. We observed that the interaction partner of Kin1 and Kin2, the t-SNARE Sec9, undergoes a size shift upon transient overexpression of the catalytically active Kin2 kinase (). Overexpression of Kin1 gave a similar shift in Sec9 mobility but induction of the kinase-dead mutants of Kin1 and Kin2 failed to induce any detectable shift in Sec9 (our unpublished data). To test whether this shift is indeed the result of phosphorylation, we analyzed the effect of phosphatase treatment on the mobility of immunoprecipitated Sec9 protein after Kin2 induction. Cells carrying either vector alone or CEN/GAL KIN2 were incubated in galactose-containing medium for 4 h to induce Kin2 expression, and lysates obtained from these cells were subjected to immunoprecipitation with anti-Sec9 antibodies. Sec9-containing immune complexes were subsequently treated with two different phosphatases: λ-phosphatase or CIP. Phosphatase treatment, but not mock control treatment, abolished the Kin2-dependent size shift of Sec9, demonstrating that the change in Sec9 mobility in response to Kin2 induction is indeed due to phosphorylation ().
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 (more ...)
We next examined whether Sec9 is phosphorylated directly by Kin1/Kin2 in vitro. We made use of recombinant Sec9 protein as a substrate in an in vitro kinase reaction and looked for [γ-32
P]ATP incorporation in the presence of immunoprecipitated Kin1/Kin2 proteins bound to protein A-Sepharose beads. The data shown represent phosphorylation reactions in the presence of Kin2; however, we found virtually identical results for immunoprecipitated Kin1 in these assays. The recombinant Sec9 protein was initially divided into three domains, Sec9-NT1 (amino acids 1-168), Sec9-NT2 (amino acids 166-401), and Sec9-CT (corresponding to the SNAP25 domain amino acids 401-651), each of which were fused to GST, expressed in bacteria, and purified as described previously (Rossi et al., 1997
). This analysis demonstrated that the Sec9-NT2 protein turned out to be an excellent substrate for Kin2 (Figure , 1), with phosphoacceptor activity significantly greater than that of casein, which was previously identified as a test substrate of Kin1 and Kin2 in vitro (Lamb et al., 1991
; Donovan et al., 1994
). We subsequently mapped the site of Sec9 phosphorylation by Kin2 to serine 315 by sequential deletion and mutagenesis of serine or threonine residues to alanine. In particular, we found that the substitution of serine 315 to alanine abolished the ability of Kin2 to phosphorylate Sec9-NT2 in vitro (Figure , 2). We used the same strategy to map a significantly weaker in vitro phosphoacceptor site in the SNAP-25 domain of Sec9 to serine 632 (our unpublished data). We next determined the effect of the mutation of these sites on the Kin2-induced phosphorylation of Sec9 in vivo. Surprisingly, we found that the Sec9-S315A as well as Sec9-S315A, S632A proteins were identical to wild-type Sec9 in the Kin2 induced phosphorylation as judged by a mobility shift (). Therefore, the major phosphoacceptor sites on Sec9 phosphorylated by Kin2 in vitro are not responsible for Kin2-induced phosphorylation of Sec9 in vivo. This result indicates that in vivo Kin1/Kin2 are unlikely to directly phosphorylate Sec9, but rather this phosphorylation occurs as a downstream effect of Kin1/Kin2 induction, presumably by direct or indirect activation of a kinase, which in turn phosphorylates Sec9 at a site or sites other than serine 315.
To assess the specificity of the catalytic activity of Kin1 and Kin2, we examined whether other components of the late exocytic machinery are phosphorylated by these kinases. In vivo experiments were performed on cells carrying either vector alone or CEN/GAL KIN2, radioactively labeled with [32P]orthophosphate during a 4-h induction of Kin2 in galactose-containing medium. Lysates from these cells were immunoprecipitated with antibodies against Sec9, Sso1/2, Sro7, and a number of components of the Exocyst complex and a subset of small GTPases involved in secretion. An identical set of strains was simultaneously labeled with [35S]methionine to control for the presence of the proteins in the lysates examined. Proteins were separated on SDS-PAGE, and their phosphorylation state was determined by autoradiography. Out of all proteins tested, only Sec9 displayed an increased level of [32P]orthophosphate incorporation in cells overexpressing Kin2 (). Thus, Kin2 specifically induces the phosphorylation of Sec9. These data allow us to hypothesize that Kin1 and Kin2 act in the secretory pathway by regulating the phosphorylation of the t-SNARE Sec9.
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. (more ...)
To address the functional significance of the Sec9 phosphorylation induced by Kin1 and Kin2, we tested whether expression of these kinases affects the subcellular localization of Sec9. We analyzed the distribution of Sec9 into pellet and supernatant fractions after centrifugation at 30,000 × g in galactose-induced and uninduced cells carrying CEN/GAL KIN1. In uninduced cells (as well as in cells carrying empty CEN/GAL vector; our unpublished data) ~50-60% of Sec9 is cytosolic and partitions into the supernatant fraction, whereas the rest is membrane bound (the Triton-sensitive pellet fraction) (). Interestingly, in cells expressing CEN/GAL KIN1 the proportion of the cytosolic Sec9 is increased relative to the membrane-bound Sec9 (). Averaging three independent experiments, induction of Kin1 expression resulted in reproducible elevation of the cytosolic Sec9 levels to ~70-75% of the total Sec9 pool. As expected, Kin1 does not alter distribution of Sso1/2 under identical conditions (). Furthermore, Sec9 undergoes a Kin1-mediated mobility shift exclusively in the cytosolic but not the membrane fraction. As expected, induction of KIN2 had the same effect on Sec9 distribution (our unpublished data). Thus, overexpression of Kin1 or Kin2 results in release of a fraction of Sec9 from the plasma membrane into the cytosol.
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 (more ...)
To determine the effect of GAL-induced Kin1 overexpression on overall growth and secretory function, we examined the ability of sec1-1 cells transformed with a GAL-KIN1 construct to grow and secrete the periplasmic protein BglII. As shown in , we find that galactose-induced expression of Kin1 protein results in dramatic suppression of the growth defect at the nonpermissive temperature of 33°C. Suppression by GAL-KIN1 is lost when the cells are grown on noninducing YPD media. We examined the ability of galactose induced sec1-1 transformants to secrete BglII after a shift to restrictive temperature. As shown in , we find that control sec1-1 cells containing an empty GAL vector show an accumulation of internal BglII. In contrast cells containing GAL-KIN1 show a dramatic suppression of the secretory defect to levels of internal BglII found in wild-type yeast cells. Therefore, the same conditions of Kin1 overexpression that result in a reduction of Sec9 levels on the membrane also yield an overall “gain of function” in the secretory pathway.
Based on these data, we suggest that the positive effect of Kin1 and Kin2 on the secretory pathway may be mediated through regulation of the plasma membrane t-SNARE Sec9. Consistent with this assumption, overexpression of SEC9
suppresses growth defects of a number of late sec
mutants that also are suppressed by introduction of multicopy KIN1
. High copy SEC9
is known to restore the viability of rho3
Δ, sec4-P48, sec1-1, sec3-2, sec8-9, sec9-4
, and sec15-1
(Lehman et al., 1999
). When we compared the relative suppression capabilities of high copy KIN1, KIN2
, and SEC9
, we found that KIN1
largely mimic the SEC9
suppression profile and exert an effect either equal or less potent in comparison with that of SEC9
(). The only mutant that KIN1
, but not SEC9
, are capable of suppressing is sec2-41
, the significance of which remains to be addressed. Together, our results are consistent with Kin1 and Kin2 acting in the secretory pathway by positively regulating Sec9 function.