From numerous individual examples, it is clear that the function of a protein can be controlled through mechanisms regulating its subcellular distribution; however, the extent to which this form of regulation occurs has not been investigated on a large scale. Accordingly, we undertook the first systematic analysis of differential protein localization for any protein set in any eukaryote, and by using the yeast kinome as the subject, we identified an interdependent subnetwork of proteins whose intracellular localization is tightly regulated during filamentous growth. This study, therefore, describes an underappreciated type of regulatory network, yields insight into the degree to which differential protein localization serves as a widespread regulatory mechanism, and also identifies the kinase Ksp1p as a new filamentous growth gene.
The plasmid collection described here constitutes a unique and versatile resource for the yeast scientific community, in complement to a very useful set of chromosomally integrated green fluorescent protein-fusions presented in Huh et al. (2003)
. Obviously, however, our plasmid collection is not without its limitations; carboxy-terminal fusions may perturb the functions of some proteins, and this is an important consideration in using these plasmids. For example, carboxy-terminal tagging is typically problematic in analyzing isoprenylated gene products and geranylgeranylated proteins (Bhattacharya et al., 1995
), as well as proteins modified with palmitoyl and farnesyl groups (Sun et al., 2004
; Roth et al., 2006
). Although some proteins of the cell wall, endoplasmic reticulum, and peroxisomes may be adversely affected by carboxy-terminal modification, we do expect the majority of yeast proteins to function normally as C-terminal fluorescent protein fusions (Pelham et al., 1988
). Plus, the alternative approach of amino-terminal tagging poses greater potential for protein mislocalization.
By live cell imaging of kinase-YFP chimeras, we found Bcy1p, Fus3p, Ksp1p, Kss1p, Sks1p, and Tpk2p differentially localized to the nucleus under filamentous growth conditions. These kinases have been characterized to varying degrees, and the pathway context of each is presented in , along with a summary of our localization data. In many cases, the observed localization shifts can be reconciled easily with corresponding protein functions. Specifically, PKA phosphorylates nuclear proteins, such as the filamentous growth transcription factor Flo8p (Rupp et al., 1999
), under filamentous growth conditions; the nuclear shift of Bcy1p and Tpk2p likely enables PKA to selectively phosphorylate nuclear-localized targets during filamentation. The filamentous growth MAPK Kss1p phosphorylates an incompletely defined set of nuclear proteins, including the transcriptional activator Ste12p. Kss1p does not shift its localization in response to mating factor (Ma et al., 1995
), and this filamentous growth-induced shift may constitute one mechanism ensuring Kss1p signaling specificity.
Figure 7. Subcellular localization of the yeast kinome during filamentous growth. This diagram summarizes the localization of 125 protein kinases, constituting the yeast kinome, under conditions of filamentous growth. In total, 119 kinases do not shift localization (more ...)
In regard to this study, it should be noted that filamentous growth can be induced by conditions of nitrogen deprivation as well as by growth in the presence of short-chain alcohols, such as butanol. Classically, pseudohyphal growth refers to a form of filamentous growth induced in diploid yeast by conditions of nitrogen deprivation on solid medium, wherein the yeast strain exhibits both surface-spread filamentation and invasive growth (Gimeno et al., 1992
; Gancedo, 2001
). Haploid strains of yeast undergo invasive growth on rich medium, but do not exhibit extensive surface-spread filamentation (Roberts and Fink, 1994
). Growth in butanol can be used to induce filamentation in haploid and diploid yeast, yielding morphological properties resembling pseudohyphal growth, even in liquid medium (Lorenz et al., 2000
). Compared with nitrogen deprivation, butanol induction involves several underlying genetic differences; for example, Lorenz et al. (2000)
report that numerous upstream nutrient-sensing genes required for classic pseudohyphal growth are not required for butanol-induced filamentous growth. We, however, identified at least two of these genes (GPA2
) in a disruption screen for genes essential in butanol-induced haploid filamentous growth (Jin et al., 2008
); thus, further analysis will be required to understand the genetic basis of these induction mechanisms. To consider both induction schemes, in this study, we assayed kinase localizations in diploid yeast under conditions of nitrogen stress and in haploid yeast by growth in butanol. Furthermore, we have endeavored to make clear the growth conditions used in each study throughout this text.
As indicated from our data, Ksp1p represents an important new filamentous growth gene, because its deletion inhibits all characteristic filamentous growth landmarks in haploid yeast: cell elongation, surface-spread filamentation, and invasive growth. Furthermore, the localization shift of Ksp1p is required for filamentous growth, as is its kinase activity. This strongly suggests that Ksp1 phosphorylates one or more nuclear proteins as an essential step in the yeast filamentous growth response. At present, Ksp1p has no confirmed targets. In vitro phosphorylation studies using protein microarrays identify 187 putative substrates for Ksp1p (Ptacek et al., 2005
); however, none of these putative substrates belong to known filamentous growth pathways. The nuclear shift of Ksp1p requires the presence of BCY1
, and SKS1
, and, at minimum, the kinase activity of Fus3p. Ksp1p is not an established target of these kinases, and the effect of Fus3p phosphorylation may be indirect. Interestingly, Ksp1p is a target for Hsf1p, Pho85p, and Pcl1p; hence, its predicted involvement in the cell cycle and in the yeast general stress response (Dephoure et al., 2005
; Hashikawa et al., 2006
). Because filamentous growth is coordinated with the cell cycle and general stress response machinery, Ksp1p may play an important role in the signaling link between these processes.
The nuclear shifts of Fus3p and Sks1p are surprising. Fus3p is phosphorylated by Ste7p and translocates to the nucleus in response to mating pheromone, where it phosphorylates Ste12p and the filamentous growth transcription factor Tec1p. In the latter case, phosphorylation of Tec1p targets it for degradation, thereby inhibiting the filamentous growth pathway during mating (Elion et al., 1993
; Choi et al., 1999
; Bao et al., 2004
). During filamentous growth, the function of Fus3p in the nucleus is unclear, because it presumably cannot phosphorylate Tec1p under these conditions. Fus3p may still be phosphorylated by Ste7p, and it may still phosphorylate Ste12p during filamentous growth. Fus3p kinase activity is required for the nuclear translocation of Ksp1p and Sks1p during filamentation, suggesting that it possesses previously unappreciated roles in the yeast filamentous growth response. The role of Sks1p in filamentous growth is also unclear, because its known functions are apparently distinct from those mediating filamentous growth, and its deletion does not affect filamentation. In this context, kinase deletion phenotypes must be interpreted with caution, because kinases are known to engage in significant cross talk and compensatory activity (Madhani and Fink, 1997
; McClean et al., 2007
). Thus, the phenotype of a deleted kinase may be masked by activity from another kinase not normally functioning in a given process.
The observed localization shifts raise an interesting question regarding the mechanism by which these kinases translocate into the nucleus. Little has been reported describing nuclear localization signals (NLSs) in these proteins, and sequence analysis by the program PSORTII (Nakai and Horton, 1999
) suggests the presence of a putative NLS in only Ksp1p. Thus, the remaining kinases are likely ferried into the nucleus through interaction with another protein. For example, under conditions of temperature stress in S288c, Bcy1p translocates from the nucleus to the cytoplasm through an interaction with the protein Zds1p (Griffioen et al., 2001
). Similar interactions with other proteins may allow for the filamentous growth-induced translocation of the kinase subset reported here, although no obvious candidates are evident from interaction data sets.
It is tempting to extrapolate our findings over the yeast proteome as a whole; however, we expect that the kinome is particularly subject to this form of regulatory control and that the overall percentage of yeast proteins regulated by differential localization will be less than the 5% rate (6 of 125) observed here. We do expect the transcription factor complement in yeast to be similarly regulated by subcellular localization to a high degree, and, in complement to this study, it would be interesting to screen the full set of yeast transcription factors for differential localization during filamentous growth.
Collectively, this study presents the first large-scale analysis of differential protein localization in any eukaryote, and the kinase network identified here defines a previously overlooked mechanism of regulatory control during yeast cell growth and development. Conceptually, regulated protein localization provides a level of specificity to otherwise promiscuous kinase activities, and the network-based structure of this regulated localization confers greater specificity still. The implications of these findings extend beyond our understanding of yeast cell biology. Similar mechanisms are assuredly at play in higher eukaryotes as well, and subsequent investigations in higher organisms will clarify the degree to which these localization-based regulatory networks are evolutionarily conserved.