A Small Conserved Domain in the Yeast Spa2p Is Necessary and Sufficient for Its Polarized Localization

SPA2 encodes a yeast protein that is one of the first proteins to localize to sites of polarized growth, such as the shmoo tip and the incipient bud. The dynamics and requirements for Spa2p localization in living cells are examined using Spa2p green fluorescent protein fusions. Spa2p localizes to one edge of unbudded cells and subsequently is observable in the bud tip. Finally, during cytokinesis Spa2p is present as a ring at the mother–daughter bud neck. The bud emergence mutants bem1 and bem2 and mutants defective in the septins do not affect Spa2p localization to the bud tip. Strikingly, a small domain of Spa2p comprised of 150 amino acids is necessary and sufficient for localization to sites of polarized growth. This localization domain and the amino terminus of Spa2p are essential for its function in mating. Searching the yeast genome database revealed a previously uncharacterized protein which we name, Sph1p (Spa2p homolog), with significant homology to the localization domain and amino terminus of Spa2p. This protein also localizes to sites of polarized growth in budding and mating cells. SPH1, which is similar to SPA2, is required for bipolar budding and plays a role in shmoo formation. Overexpression of either Spa2p or Sph1p can block the localization of either protein fused to green fluorescent protein, suggesting that both Spa2p and Sph1p bind to and are localized by the same component. The identification of a 150–amino acid domain necessary and sufficient for localization of Spa2p to sites of polarized growth and the existence of this domain in another yeast protein Sph1p suggest that the early localization of these proteins may be mediated by a receptor that recognizes this small domain.Polarized cell growth and division are essential cellular processes that play a crucial role in the development of eukaryotic organisms. Cell fate can be determined by cell asymmetry during cell division (Horvitz and Herskowitz, 1992; Cohen and Hyman, 1994; Rhyu and Knoblich, 1995). Consequently, the molecules involved in the generation and maintenance of cell asymmetry are important in the process of cell fate determination. Polarized growth can occur in response to external signals such as growth towards a nutrient (Rodriguez-Boulan and Nelson, 1989; Eaton and Simons, 1995) or hormone (Jackson and Hartwell, 1990a , b ; Segall, 1993; Keynes and Cook, 1995) and in response to internal signals as in Caenorhabditis elegans (Goldstein et al., 1993; Kimble, 1994; Priess, 1994) and Drosophila melanogaster (St Johnston and Nusslein-Volhard, 1992; Anderson, 1995) early development. Saccharomyces cerevisiae undergo polarized growth towards an external cue during mating and to an internal cue during budding. Polarization towards a mating partner (shmoo formation) and towards a new bud site requires a number of proteins (Chenevert, 1994; Chant, 1996; Drubin and Nelson, 1996). Many of these proteins are necessary for both processes and are localized to sites of polarized growth, identified by the insertion of new cell wall material (Tkacz and Lampen, 1972; Farkas et al., 1974; Lew and Reed, 1993) to the shmoo tip, bud tip, and mother–daughter bud neck. In yeast, proteins localized to growth sites include cytoskeletal proteins (Adams and Pringle, 1984; Kilmartin and Adams, 1984; Ford, S.K., and J.R. Pringle. 1986. Yeast. 2:S114; Drubin et al., 1988; Snyder, 1989; Snyder et al., 1991; Amatruda and Cooper, 1992; Lew and Reed, 1993; Waddle et al., 1996), neck filament components (septins) (Byers and Goetsch, 1976; Kim et al., 1991; Ford and Pringle, 1991; Haarer and Pringle, 1987; Longtine et al., 1996), motor proteins (Lillie and Brown, 1994), G-proteins (Ziman, 1993; Yamochi et al., 1994; Qadota et al., 1996), and two membrane proteins (Halme et al., 1996; Roemer et al., 1996; Qadota et al., 1996). Septins, actin, and actin-associated proteins localize early in the cell cycle, before a bud or shmoo tip is recognizable. How this group of proteins is localized to and maintained at sites of cell growth remains unclear.Spa2p is one of the first proteins involved in bud formation to localize to the incipient bud site before a bud is recognizable (Snyder, 1989; Snyder et al., 1991; Chant, 1996). Spa2p has been localized to where a new bud will form at approximately the same time as actin patches concentrate at this region (Snyder et al., 1991). An understanding of how Spa2p localizes to incipient bud sites will shed light on the very early stages of cell polarization. Later in the cell cycle, Spa2p is also found at the mother–daughter bud neck in cells undergoing cytokinesis. Spa2p, a nonessential protein, has been shown to be involved in bud site selection (Snyder, 1989; Zahner et al., 1996), shmoo formation (Gehrung and Snyder, 1990), and mating (Gehrung and Snyder, 1990; Chenevert et al., 1994; Yorihuzi and Ohsumi, 1994; Dorer et al., 1995). Genetic studies also suggest that Spa2p has a role in cytokinesis (Flescher et al., 1993), yet little is known about how this protein is localized to sites of polarized growth.We have used Spa2p green fluorescent protein (GFP)¹ fusions to investigate the early localization of Spa2p to sites of polarized growth in living cells. Our results demonstrate that a small domain of ∼150 amino acids of this large 1,466-residue protein is sufficient for targeting to sites of polarized growth and is necessary for Spa2p function. Furthermore, we have identified and characterized a novel yeast protein, Sph1p, which has homology to both the Spa2p amino terminus and the Spa2p localization domain. Sph1p localizes to similar regions of polarized growth and sph1 mutants have similar phenotypes as spa2 mutants.     

Analysis of Free Energy Signals Arising from Nucleotide Hybridization Between rRNA and mRNA Sequences during Translation in Eubacteria

A decoding algorithm is tested that mechanistically models the progressive alignments that arise as the mRNA moves past the rRNA tail during translation elongation. Each of these alignments provides an opportunity for hybridization between the single-stranded, -terminal nucleotides of the 16S rRNA and the spatially accessible window of mRNA sequence, from which a free energy value can be calculated. Using this algorithm we show that a periodic, energetic pattern of frequency 1/3 is revealed. This periodic signal exists in the majority of coding regions of eubacterial genes, but not in the non-coding regions encoding the 16S and 23S rRNAs. Signal analysis reveals that the population of coding regions of each bacterial species has a mean phase that is correlated in a statistically significant way with species () content. These results suggest that the periodic signal could function as a synchronization signal for the maintenance of reading frame and that codon usage provides a mechanism for manipulation of signal phase.[1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29,30,31,32]     

Splitting the BLOSUM Score into Numbers of Biological Significance

Mathematical tools developed in the context of Shannon information theory were used to analyze the meaning of the BLOSUM score, which was split into three components termed as the BLOSUM spectrum (or BLOSpectrum). These relate respectively to the sequence convergence (the stochastic similarity of the two protein sequences), to the background frequency divergence (typicality of the amino acid probability distribution in each sequence), and to the target frequency divergence (compliance of the amino acid variations between the two sequences to the protein model implicit in the BLOCKS database). This treatment sharpens the protein sequence comparison, providing a rationale for the biological significance of the obtained score, and helps to identify weakly related sequences. Moreover, the BLOSpectrum can guide the choice of the most appropriate scoring matrix, tailoring it to the evolutionary divergence associated with the two sequences, or indicate if a compositionally adjusted matrix could perform better.[1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29]     

An Export-Specific Reporter Designed for Gram-Positive Bacteria: Application to Lactococcus lactis

The identification of exported proteins by fusion studies, while well developed for gram-negative bacteria, is limited for gram-positive bacteria, in part due to drawbacks of available export reporters. In this work, we demonstrate the export specificity and use of the Staphylococcus aureus secreted nuclease (Nuc) as a reporter for gram-positive bacteria. Nuc devoid of its export signal (called Δ_SPNuc) was used to create two fusions whose locations could be differentiated. Nuclease activity was shown to require an extracellular location in Lactococcus lactis, thus demonstrating the suitability of Δ_SPNuc to report protein export. The shuttle vector pFUN was designed to construct Δ_SPNuc translational fusions whose expression signals are provided by inserted DNA. The capacity of Δ_SPNuc to reveal and identify exported proteins was tested by generating an L. lactis genomic library in pFUN and by screening for Nuc activity directly in L. lactis. All Δ_SPNuc fusions displaying a strong Nuc⁺ phenotype contained a classical or a lipoprotein-type signal peptide or single or multiple transmembrane stretches. The function of some of the predicted signals was confirmed by cell fractionation studies. The fusions analyzed included long (up to 455-amino-acid) segments of the exported proteins, all previously unknown in L. lactis. Homology searches indicate that several of them may be implicated in different cell surface functions, such as nutrient uptake, peptidoglycan assembly, environmental sensing, and protein folding. Our results with L. lactis show that Δ_SPNuc is well suited to report both protein export and membrane protein topology.Most exported proteins are targeted for transport by a primary export signal comprising a hydrophobic domain. The signal can be present at the protein N terminus and cleaved during transport (i.e., signal peptide), but it can also remain embedded in the membrane (i.e., transmembrane segment) (63). Exported proteins are estimated to represent about 20% of total cellular proteins in gram-negative bacteria (39, 44), and contribute to various essential processes like nutrient uptake, macromolecular transport and assembly, envelope biogenesis and integrity, motility, cell division, energy generation, scavenging and detoxification, signal transduction, stress resistance, cell communication, and virulence in the case of pathogens.Several years ago, the elegant strategy of translational fusion to an export-specific reporter protein was designed to specifically isolate genes encoding exported proteins. This kind of reporter is translocation competent but unable to direct its own export (it corresponds to a signal peptideless form of an exported protein), and its activity requires an extracytoplasmic location. Among a library of proteins N-terminally fused to such a reporter, only fusions having the proper signal are exported and active. This strategy was first described for Escherichia coli using alkaline phosphatase (PhoA) as a reporter (16, 36); since then it has been applied to many gram-negative bacteria, particularly pathogens (for reviews, see references 24 and 35 and references therein).Export-specific reporters have a potentially important use in gram-positive bacteria, not only for protein identification and structural analyses, but also for technological applications. Most studies directly adopted the gram-negative reporters available, PhoA and the E. coli TEM β-lactamase (BlaM) (5). The Bacillus licheniformis α-amylase, AmyL, has also been used (17). Surprisingly, relatively few fusion studies allowed identification and characterization of the exported proteins (32, 42). In many cases, only the export signal was characterized (17, 18, 43, 51, 54, 55), possibly because only very short polypeptides (60 amino acids) were fused to the reporter.The rather limited results obtained by using reporter fusions may reveal that the reporters used are not fully adapted for use in gram-positive bacteria. (i) Fusions to gram-negative reporters PhoA and BlaM seem to display little activity and/or to be less stable in gram-positive bacteria, probably because of improper folding (42, 54). Both PhoA (active as a dimer) and BlaM folding require disulfide bond formation, which is catalyzed by DsbA in various gram-negative bacteria (3, 22); it is not yet clear whether such a process exists in gram-positive bacteria (19). Furthermore, altered codon usage and GC content may decrease expression of reporter genes. (ii) Selection of BlaM fusions has been routinely performed in E. coli, possibly due to difficulties of direct ampicillin resistance selection in gram-positive bacteria (43, 51, 54). Such preselection may create a bias due to species specificity of export signals, which, for signal peptides, are significantly longer in gram-positive bacteria (65). (iii) AmyL, a reporter of gram-positive origin, may be the best suited for use in gram-positive bacteria. However, the plate detection test results in loss of cell viability (18a), and thus its use requires replica plating (17, 18).The above-mentioned considerations led us to design a protein export reporter which would be suitable for use in a broad host range of gram-positive bacteria. The reporter we chose is based on the Staphylococcus aureus secreted nuclease (Nuc), a small, stable, monomeric, extensively studied enzyme (EC 3.1.31.1 [9]), having a mature form devoid of cysteine residues (50). Nuc is efficiently secreted by various gram-positive bacteria as an active 168-amino-acid polypeptide which may undergo subsequent proteolytic cleavage of the N-terminal 19- to 21-amino-acid propeptide to give rise to another active form, called NucA (27, 30, 31, 38, 58). The enzymatic activity test for Nuc is sensitive and nontoxic to colonies (28, 29, 50). Several features of Nuc thus make it a potentially optimal candidate for reporting protein export in gram-positive bacteria.In this study, we show that a truncated form of Nuc lacking its export signal (called Δ_SPNuc) is an export-specific reporter. A shuttle vector, pFUN (for fusion to nuclease), was designed to specifically identify genes encoding exported proteins as translational fusions to Δ_SPNuc. pFUN was developed and used to study protein export in Lactococcus lactis, a gram-positive microaerophilic industrial microorganism used in dairy fermentations (37). Despite the technological importance of surface and extracellular proteins in this organism, export of relatively few proteins (excluding plasmid- or transposon-encoded proteins) has been reported to date (4, 6, 12, 13, 15, 26, 40, 60–62). In this work, we characterize 16 previously unknown exported L. lactis proteins. Our results confirm that Δ_SPNuc is a sensitive and specific export reporter for L. lactis and potentially for other gram-positive bacteria.     

Algorithms for Finding Small Attractors in Boolean Networks

A Boolean network is a model used to study the interactions between different genes in genetic regulatory networks. In this paper, we present several algorithms using gene ordering and feedback vertex sets to identify singleton attractors and small attractors in Boolean networks. We analyze the average case time complexities of some of the proposed algorithms. For instance, it is shown that the outdegree-based ordering algorithm for finding singleton attractors works in time for , which is much faster than the naive time algorithm, where is the number of genes and is the maximum indegree. We performed extensive computational experiments on these algorithms, which resulted in good agreement with theoretical results. In contrast, we give a simple and complete proof for showing that finding an attractor with the shortest period is NP-hard.[1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29,30,31,32]