BIOLOGICAL OVERVIEW

How do two cells, the progeny from a single cell division, develop different fates? This is the fundamental question of developmental biology. prospero and numb provide clues to one possible mechanism.

When Prospero and Numb proteins are first made, they congregate in the cell's cytoplasm, but unlike many proteins they are not distributed uniformly throughout the cell. Rather, they form a cresent pattern at one end of the cell, just under the cell membrane, between the membrane and the cell's centrioles (Hirota, 1995, Spana, 1995a and 1995b, and Knoblich, 1995). The cell is said to be polarized; that is, one end is somehow different from the other end.

Is the centriole the immediate cause of Prospero and Numb localization? One might consider that the two centrioles are not identical, and that one directs Prospero and Numb localization? But there are other elements involved in cell polarity, and their involvement must be considered. The cytoskeleton and the cell membrane are also involved. Perhaps the cell membrane acts as a tether for Prospero and Numb. In fact, the region directly under the cell membrane, the cortex is known to be highly structured with subcortical cytoskeletal elements.

Action of Inscuteable, a cytoskeleton associated protein, suggests an involvement of cytoskeleton in providing cues for Prospero and Numb localization. Inscuteable localizes opposite the pole to which Prospero and Numb localize. In inscuteable mutants both Prospero and Numb form cresents, but the position of both the spindle and the position of Prospero and Numb is disrupted, that is, Prospero and Numb do not localize preferentially localize. Cytochalasin D, which disrupts Actin filaments, eliminates Inscuteable cresents and results in incorrect Prospero crescent positioning, while treatment with colcemid, which destroys microtubules (and consequently the spindle) does not effect Inscuteable localization (Kraut, 1996).

In yeast the site of the previous bud determines the cell polarity. Does the same thing happen in the fly? It seems not. Instead Inscuteable localizes to the apical cortex of ectodermal cells and neuroblasts (the apical direction is towards the surface and is opposite to the basal pole), irrespective of the orientation of their previous division, suggesting that the localization responds to the apical-basal polarity.

It appears that there must be an organizer to provide positional information for both the orientation of the mitotic spindle and asymmetric localization of Numb and Prospero (Knoblich, 1995). The dynamics of Inscuteable suggests that the microfilamental network, organized with respect to apical-basal polarity, acts to organize both the Numb and Prospero by acting through Inscuteable. Likewise Inscuteable acts to organize the microtubule network and thus the plane of cell division. For more information an apical-basal polarity, see the Crumbs site.

Relevant to this discussion is a structure found oogenesis called the fusome. Fusomes consist of cytoskeletal proteins, alpha-Spectrin, ß-Spectrin, Hu-li tai shao (an adducin-like protein) and Ankyrin. Of particular interest is the association of fusomes with the pole of the mitotic spindle (Lin, 1995). During the first cystoblast (cystoblasts are derived from germ line stem cells) division, fusome material is associated with only one pole of the mitotic spindle, revealing that this division is asymmetric. During the subsequent three divisions, the growing fusome always associates with the pole of each mitotic spindle that remains in the mother cell, and only extends through the newly formed ring canals after each division is completed. The association of fusome proteins with the mitotic spindle indicates a direct interaction between cytoskeletal components and only one pole of the mitotic spindle. Surely this must have something to do with the underlying mechanism of asymmetric cell division.

As a cell divides, Prospero and Numb are asymmetrically distributed to the two progeny. The cell receiving the two proteins has a different fate from that of its sister. In the cell receiving both Prospero and Numb ( the ganglion mother cell), Prospero migrates from its cytoplasmic position into the nucleus, where they assumes the role of transcription factor, thus establishing the neural fate of the cell in which it is found. Numb function is to antagonize Notch signaling, and has been shown to directly interact with the cytoplasmic domain of Notch (Guo, 1996). The second progeny, the one that does not receive Prospero or Numb, is in effect, a new neuroblast. This cell will continue to divide, generating new ganglion mother cells and new neuroblasts (Knoblich, 1995, Rhyu, 1995 and Spana, 1995) A special sequence in Numb and Prospero protein is responsible for localization of the protein in the cortex, immediately adjacent to the cell membrane (Hirata, 1995). With what protein does Numb and Prospero interact? An answer to this question will still not shed light on how apical-basal polarity is established, but it will help in understanding how Prospero and Numb become asymmetrically distributed.

A similar asymmetrical system of Prospero distribution operates in other cells expressing prospero, such as the precursors of glia (Spana, 1995).

Specification of cell fate in the adult sensory organs is known to be dependent on intrinsic and extrinsic signals. Prospero (Pros) acts as an intrinsic signal for the specification of cell fates within the mechanosensory lineage. The sensory organ precursors divide to give rise to two secondary progenitors: PIIa and PIIb. Pros is expressed in PIIb, which gives rise to the neuron and thecogen cells. Pros expression was first detected among the secondary progenitors in the nucleus of the more anteriorly located cell (PIIb). Interestingly, this was the first cell to divide in all cases examined. Prior to cell division, Pros becomes uniformly distributed on the cortical membrane and throughout the cytosol. Unlike the findings in the central nervous system, Pros is not asymmetrically localized in cells at any stage of the lineage. Asymmetric crescents of Pros immunoreactivity are not observed even after blocking mitosis with colcemid. These data suggest that Pros is expressed first in the nucleus and then generally in the cytosol of PIIb (Reddy, 1999).

Loss of Notch function generated using either a conditional mutant allele or by misexpressing Numb protein results in the ectopic expression of Pros in PIIa. This observation is consistent with a PIIa-to-PIIb conversion by Notch loss of function or nb gain of function. It is not clear whether the apparent negative regulation of Pros by Notch is a direct effect or merely reflects the altered fate of the cells that is caused by other molecular factors. Pros misexpression is sufficient for the transformation of PIIa to PIIb fate (Reddy, 1999).

Pros is not asymmetrically localized in PIIb; following division of this cell, the protein is detected in both the progeny. This is strikingly different from findings in the CNS where Pros, together with Numb and Miranda, is localized asymmetrically in the neuroblasts and inherited by the GMC. Following division of PIIb, Pros is inherited by nuclei of both progeny. Expression in one of the siblings decays rapidly and this cell differentiates as the neuron. Pros immunoreactivity is sustained in the thecogen cell possibly due to de novo synthesis. The requirement for pros function in PIIb precludes analysis of its later role after division. In experiments where Pros was misexpressed in all four cells of the sensory organ, a conversion of external to internal cells is observed, consistent with a PIIa-to-PIIb transformation. Neuronal cells form normally despite the fact that they express the thecogen cell marker. Similarly, Notch loss of function and Numb gain of function results in a conversion of all four cells of the lineage to neurons. Pros is expressed in all these cells under these conditions. These observations together demonstrate that pros activity is not sufficient for identity of the thecogen cell and that neuronal cell differentiation can occur normally despite Pros expression. The elucidation of pros function in the thecogen cell awaits the availability of a hypomorphic mutant allele that can allow loss of function in the thecogen cell without affecting the secondary progenitors. Pros has been shown to be expressed in several CNS- as well as PNS-associated glial cells and it is possible that it plays a role in the later differentiation and/or function of these cells (Reddy, 1999).

In the normal mechanosensory lineage, Notch is involved in the binary choice between thecogen and neuron. In this lineage Notch signaling is experienced by the cell that ultimately becomes the thecogen cell. This is distinct from the scenario at the secondary progenitor stage, where the cell that experiences Notch signaling does not express Pros. This means that, unlike in PIIb, Notch signaling does not downregulate Pros in the thecogen cell. There are several possible explanations for this finding. One possibility is that Pros protein is merely partitioned to the daughters of the PIIb after division. It therefore serves no role in the binary choice of thecogen versus neuron but is synthesized de novo after these cells have acquired their identity. At this later time point, the thecogen cell is no longer experiencing the Notch signal. Another possibility lies in the different effector mechanisms utilized for Notch activity. In three instances (during lateral signaling, PIIa-PIIb choice, as well as in the PIIa lineage) Notch activation results in release of Su(H) from its binding site on the cytoplasmic domain of Notch and its translocation to the nucleus. Su(H) protein can be detected in the nucleus of the socket cell; however, the role of Su(H) cannot be seen in the PIIb lineage. Extra copies of Su(H) do not produce thecogen-to-neuron transformations, suggesting that Notch signaling in the thecogen/neuron choice occurs by a Su(H)-independent mechanism. Thus the regulation of Pros expression could be mediated by Notch through a Su(H)-independent event (Reddy, 1999 and references).

Regulated transcription of the prospero gene in the Drosophila eye provides a model for how gene expression is specifically controlled by signals from receptor tyrosine kinases. prospero is controlled by signals from the Egfr receptor and the Sevenless receptor. A direct link is established between Egfr activation of a transcription enhancer in prospero and binding of two transcription factors that are targets of Egfr signaling. Binding of the cell-specific Lozenge protein is also required for activation, and overlapping Lozenge protein distribution and Egfr signaling establishes expression in a subset of equivalent cells competent to respond to Sevenless. Sevenless activates prospero independent of the enhancer and involves targeted degradation of Tramtrack, a transcription repressor (Xu, 2000).

Thus, Egfr signaling is required to activate pros expression in the R7 equivalence group but is restricted from activating pros expression in other cells by the distribution of the transcription factor Lz. The transcriptional effectors of the Egfr pathway combinatorially interact with Lz at an eye-specific pros enhancer to restrict enhancer activity to the R7 equivalence group. It is suggested that this mechanism is a primary means by which pros transcription is restricted to the R7 equivalence group. This combinatorial mechanism supposes that Egfr signaling inactivates Yan and activates Pnt, but modification of these transcription factors is not sufficient to activate the enhancer. Lz is also required to activate the enhancer. The only cells that contain Lz, activated Pnt, and inactivated Yan are R1, R6, R7, and cone cells. Thus, the enhancer is activated in a subset of Egfr-responsive cells. A similar combinatorial mechanism regulates shaven expression in cone cells. shaven expression requires both Lz and Egfr-induced regulation of Yan and Pnt. However, Notch signaling through Su(H) is also required for shaven expression in cone cells. This third input may explain why shaven has a more restricted expression pattern than pros, given that cone cells receive a robust Notch signal (Flores, 2000). In muscle and cardiac cells, RTK signaling is similarly integrated with other signal inputs and tissue-restricted transcription factors to regulate enhancer activity of the even skipped gene (Halfon, 2000). Thus, differential expression of genes in response to an RTK/Ras signal appears to be controlled by each gene's capacity to bind and be regulated by different combinations of transcription factors (Xu, 2000).

A model is presented for the regulatory inputs into prospero. (1) In eye progenitor cells, the presence of Yan represses pros transcription through its binding to the enhancer and competitively excluding Pnt from binding to the same sites. (2) Lz begins to be produced in progenitor cells after the first wave of photoreceptor differentiation. However, Lz alone cannot activate the enhancer in progenitor cells that have not received a Spitz signal. (3) When a progenitor cell receives a Spitz signal, Egfr is activated. This inactivates Yan, allowing activated Pnt to bind to the enhancer. At the morphogenetic furrow, the enhancer is inactive despite Egfr-stimulated cells containing inactive Yan and active Pnt since progenitor cells in this region do not contain Lz, which is also required for enhancer activity. Hence, photoreceptors R2, R3, R4, R5, and R8 do not express pros. It is only in cells that receive a Spitz signal and contain Lz that the combination of Lz and Pnt bound to the enhancer activate the enhancer. (4) Ttk88 reduces the level of pros transcription through a mechanism independent of the eye enhancer. This repression may not be strong enough to block the eye enhancer in the R7 equivalence group but acts to limit its level of transcription. (5) When a progenitor cell receives both a Spitz and Boss signal, stronger or longer signal transduction induces Ttk88 inactivation. This Egf represses pros transcription and leads to a specific increase of Pros in R7 cells (Xu, 2000).

The ETS factors Yan and Pnt have been implicated as substrates for activated MAPK, whose activities are modified upon phosphorylation. Both Yan and Pnt bind to the same sites in the pros eye enhancer except for one site that is Yan-specific. Their effects on enhancer activity are antagonistic; Yan represses while Pnt activates. One model is that Yan represses transcription by outcompeting Pnt for their binding sites, thereby preventing Pnt from activating transcription. This model is attractive since it has been found that Yan has a 100-fold greater affinity than Pnt for ETS factor binding sites in vitro. If this difference between purified fusion proteins in vitro is extrapolated to the fly eye, it would explain how Yan can outcompete Pnt and repress transcription. Results from mutagenesis of the binding sites is also consistent with this model. Mutated binding sites cause the enhancer to be inactive, which is the result predicted if Yan merely prevents Pnt from interacting with those sites. If Yan were actively repressing transcription in a manner dependent upon binding, then mutated binding sites would cause derepression and ectopic expression. Although a model where the binding sites are obligatory for both active repression by Yan and activation by Pnt cannot be excluded, the competitive binding model is the simplest one consistent with these data (Xu, 2000).

From these data it is proposed that two RTKs, Egfr and Sev, regulate pros by activating the Ras1 intracellular pathway in R7 cells, but these RTKs regulate pros differentially. Egfr regulates pros by modifying Yan and Pnt, which act directly through the eye-specific enhancer. The Egfr signal in R7 cells appears to occur before Sev, and it sufficiently inactivates Yan and activates Pnt to switch on the enhancer before the Sev signal. This sufficiency is demonstrated in sev mutants where enhancer activity in R7 cells is no different from wild-type. In contrast, the Sev signal in R7 cells is not sufficient to switch on the enhancer in the absence of the Egfr signal since the enhancer is inactive in Egfr mutant R7 cells (Xu, 2000).

Sev regulates pros in R7 cells by inactivating Ttk88, which otherwise represses pros through sequence elements distinct from the eye-specific enhancer. This is demonstrated by finding that overproduced Ttk88 blocks Sev from activating pros, and Sev can regulate the eye-specific enhancer only if it is linked with Ttk88 binding sites. It is not clear if the Sev signal is sufficient to inactivate Ttk88 without an Egfr input since the assay for Ttk88 activity is a reporter gene that includes the eye-specific enhancer. It is quite possible that Ttk88 inactivation in R7 cells requires both Sev and Egfr signals, since Ebi, acting downstream of Egfr to promote Ttk88 degradation, and Phyl/Sina, acting downstream of Sev, are both required to inactivate Ttk88 in R7 cells (Xu, 2000).

How do these RTKs selectively regulate particular transcription factors and thereby regulate different aspects of pros transcription? The most attractive model is that RTK selection reflects the timing or intensity of each signal. If it is timing, then there must be a time period of competence during which a factor is sensitive to any RTK signal, and the time period is different for each factor. Alternatively, the intensity of a signal may dictate which transcription factor activities are sensitive. For example, Yan and Pnt activities may be insensitive to signal strength that is less than or equal to the level achieved by Sev but not Egfr within R7 cells. Ttk88 activity may be insensitive to signal strength that is less than or equal to the level achieved by Sev or Egfr alone but not the combination of the two within R7 cells. Signal 'strength' may be determined by the level of Ras pathway activity or the length of time that the Ras pathway is active. Sensitivity of transcription factors might be set either by the affinities of these factors for binding sites in a gene such as pros, or by the ability of factors to be substrates for RTK-stimulated modification. Given that Yan and Pnt are modified by a very different mechanism from Ttk88, substrate sensitivity is a possible determinant. In summary, RTK signals may provide specificity to gene regulation based on quantitative variation in which threshold transcription responses are set by transcription factors that have different sensitivities to RTK signal strength (Xu, 2000).

GENE STRUCTURE

Bases in 5' UTR - 1089

Exons - two

Bases in 3' UTR - 1097

PROTEIN STRUCTURE

Amino Acids - 1403

Structural Domains

The Prospero transcript is alternatively spliced to encode two proteins: ProsL protein (1403 amino acids, predicted 165 kDa) and ProsS protein (1374 amino acids, predicted 160 kDa). The extra 29 amino acids in ProsL are at the beginning of the homeodomain (Chu-LaGraff, 1991).

Prospero has a novel homeodomain and three PEST domains which confer susceptability to rapid degradation. There is also a CAX (opa) repeat, a common transcription activation domain (Doe, 1991 and Vaessin, 1991).

Prospero promotes neural differentiation in Drosophila, and its activity is tightly regulated by modulating its subcellular localization. Prospero is exported from the nucleus of neural precursors but imported into the nucleus of daughter cells, which is necessary for their proper differentiation. Prospero has a highly divergent putative homeodomain adjacent to a conserved Prospero domain; both are required for sequence-specific DNA binding. The structure of these two regions consists of a single structural unit (a homeo-prospero domain), in which the Prospero domain region is in position to contribute to DNA binding and also to mask a defined nuclear export signal that is within the putative homeodomain region. It is proposed that the homeo-prospero domain coordinately regulates Prospero nuclear localization and DNA binding specificity (Ryter, 2002).

The C-terminal 163 amino acids of Drosophila Prospero (residues 1241-1403) were overexpressed, purified, and crystallized. The X-ray structure was determined at 2.05 Å resolution. Despite classification as highly divergent based on sequence comparisons, residues 1241-1303 were predicted to be capable of assuming a canonical homeodomain structure. In fact, structural analysis shows that this region does assume an overall fold very similar to homeodomains but with one striking difference. In every homeodomain structure determined to date, the so-called DNA recognition helix is either at the extreme C terminus of the protein or leads into a flexible linker of variable length that connects to another essentially independent domain. In Prospero, the recognition helix (alpha₃) connects the putative homeodomain and the Prospero domain as a single structural unit. The region corresponding to the Prospero domain can be described as a four-helix bundle (alpha₃–alpha₆). Residues 1314-1326 are disordered and residues 1368-1370 and 1391-394 form short 3₁₀ helices. While interactions between the homeo- and Prospero domain regions occur primarily within the hydrophobic core, the C-terminal residues of the Prospero domain region make additional contacts with the homeodomain region by extending into a cleft between helices alpha₁ and alpha₂ (Ryter, 2002).

A structural comparison of the homeodomain region (HD) alone reveals that, while possessing a highly divergent class sequence, the structure assumes a fold most similar to the Drosophila Engrailed homeodomain. A detailed residue-by-residue analysis was possible. A majority of the core residues seen in the homeodomain region are either invariant or conserved hydrophobic with respect to the Engrailed homeodomain sequence or homeodomain consensus sequence, indicating that this region possesses a core consistent with a standard homeodomain hydrophobic core. Variations occur primarily at the loop between alpha₁ and alpha₂ and in the middle of alpha₃. In these regions, residues that would be on the surface of a typical homeodomain are replaced by bulky hydrophobic groups that interact with the hydrophobic core of the Prospero domain. Within the loop, Phe1261 and Trp1262, along with the invariant Phe1260, create a very hydrophobic turn that protrudes into this core region. In the middle of alpha₃, Phe1298, Tyr1299, Tyr1300, and Met1303 contribute to the hydrophobic core of the Prospero domain four-helix bundle (Ryter, 2002).

The Prospero domain region (PD) is composed of a four-helix bundle (alpha₃-alpha₆). Consecutive helices are antiparallel and all helices interact via ridges in grooves. The up-down-up-down topology is the simplest found among four-helix bundles and in this case is right turning. The central residues of this bundle, representing the alpha-carbons in closest proximity, are Ala1307 of alpha₃ and Phe1361 of alpha₅. The ~50° crossing angle between the helix axes is also the most common angle found in globular proteins. The core consists of hydrophobic residues throughout that are either invariant or highly conserved throughout the Prospero/Prox1 protein family. As noted above, a number of residues within the homeodomain region also contribute to this hydrophobic core (Ryter, 2002).

The homeodomain and the Prospero domain regions do not just touch each other; rather, they interact quite extensively. If the two regions are considered as separate entities, then 22% (1499 Ų) of the surface area of the homeodomain region contributes to the interface with the Prospero domain region and, vice versa, 35% of the surface of the Prospero domain region contributes to the same interface. These fractions are significantly higher than the average value of 15% that is observed for the surface area involved in the interfaces of typical protein dimers. It confirms that the homeodomain and Prospero domain regions combine to form a distinct structural unit (Ryter, 2002).

There are extensive hydrophobic core contacts that occur between the HD and PD regions. A hydrophobic cleft on the HD is formed between Trp1262 and Tyr1274 of the strand just C-terminal to alpha₁ and alpha₂, respectively. SeMet1259 forms the floor of this cleft, with Val1263 and Val1270 flanking one end. Seated in the cleft are Val1389, Pro1390, and Phe1393 of the PD region. This cleft is flanked at the opposite end by a hydrogen-bonding interaction between His1252 and Asp1277. Phe1275, of the strand C-terminal to alpha₂, positions Lys1255 of alpha₁ in an orientation optimal for a number of hydrogen-bonding interactions. These include direct interactions with backbone carbonyls of residues 1274 and 1393, thereby creating contacts between HD alpha₁ and alpha₂, as well as the PD region (Ryter, 2002).

There are interesting similarities (see 1mijA at Structural Neighbours in PDB90 and structural alignments) between the homeo-prospero domain (HPDP structure) and that of the MATa1/MATalpha2 homeodomain heterodimer bound to DNA (Li, 1998). In the HPD structure, the C terminus of the PD region (residues 1388-1396) extends into a cleft between alpha₁ (residues 1250-1259) and alpha₂ (residues 1268-1274) of the HD region. This rather lengthy segment includes a 3₁₀ helix (residues 1391-1394) and contacts the HD region on the face opposite its DNA binding surface. This interaction between HD and PD is stabilized by hydrophobic interactions, as well as a number of hydrogen bonds. In the homeodomain heterodimer complex between MATa1 and MATalpha2, the C terminus of MATalpha2 also extends away from the body of the domain and similarly makes hydrophobic and hydrogen bond interactions within a groove formed between alpha₁ and alpha₂ of MATa1. This binding groove on the homeodomain MATa1 corresponds to that within the HD region of Prospero. In addition, the polypeptide chain that binds within the respective grooves in both cases adopts an alpha-helical or 3₁₀-helical conformation (Ryter, 2002).

The MATa1/MATalpha2 homeodomain complex is required for cell type-specific transcriptional repression in yeast. The Prospero HPD is also required for regulating transcription, but may have an additional function in controlling the subcellular localization of Prospero. Demidenko (2001) utilized molecular dissection of the HPD with expression of chimeric proteins in mammalian and insect cultured cells to show that residues 1248–1261, encompassing alpha₁, contain a nuclear export signal (NES). In the absence of any portion of the PD region, Prospero is exported from the nucleus via the Exportin pathway and subsequently found in the cytoplasm, while presence of this region blocks nuclear export and allows Prospero to accumulate in the nucleus. The HPD structure strongly suggests that it is the extreme C terminus of the PD region that sterically prevents access to the nuclear export signal (Ryter, 2002).

Interestingly, previous analysis of Prospero during asymmetric cell division has shown its phosphorylation state correlates with subcellular localization. Cytoplasmic Prospero is highly phosphorylated compared to nuclear Prospero, which raises the possibility that phosphorylated HPD may have a more open conformation in which the NES is exposed (Ryter, 2002).

In a structural comparison, the structure of the HD region was shown to be most similar to the Engrailed homeodomain. It is therefore possible to use the structural superposition matrix to model the HPD structure in complex with DNA. Three regions of potential protein-DNA contact are suggested. (1) The DNA recognition helix alpha₃ appears to contact the major groove of the DNA; (2) the N-terminal arm appears to contact the minor groove of the DNA, and (3) unexpectedly, the PD region appears to contact the DNA backbone via residues within or close to the N terminus of helix alpha₆. Engrailed homeodomain residues Asn51 (Pros HD Asn1294) and Arg53 (Pros HD Arg1296) are invariant and presumably contact the DNA in a similar fashion. In the Engrailed structure, Ile47 (Pros HD Lys1290) and Lys50 (Pros HD Ser1293) also contact the bases in the major groove to provide binding site discrimination. Pros HD Glu1297 appears poised in the major groove available for possible water-mediated contacts. Additionally, there are a number of residues that are not only invariant between the Engrailed homeodomain and the Prospero HD region, but are also positioned to make similar contacts with the DNA phosphate backbone as seen in the Engrailed homeodomain structure. In particular, Engrailed homeodomain Arg53 (Pros HD Arg1296) and Tyr25 (Pros HD Tyr1265) are predicted to interact directly with one strand of the DNA backbone in a similar fashion, while Trp48 (HD Trp1291) could interact with the opposite strand (Ryter, 2002).

In the Engrailed homeodomain structure, the N-terminal arm (residues 5-9) fits into the DNA minor groove, supplementing the contacts made by the recognition helix. In the Prospero HPD-DNA model, the N-terminal arm has a somewhat different alignment but could easily move so as to contact the DNA. A sequence comparison reveals predominantly conservative differences between the Engrailed and the Prospero sequences in the N-terminal arm, except at Engrailed homeodomain Arg5 (Pros HD Ser1245), a position shown to be important for binding site discrimination (Ryter, 2002).

This structural comparison also suggests that the Prospero HD region may contact DNA as well. Lys1380, -1376, and -1375, found within or close to the N terminus of the alpha₆ helix, are all poised to potentially interact with the DNA phosphate backbone. The basic nature of this region is further manifest in the negative electrostatic potential of the molecular surface. Tyr1379 and Ser1373 could possibly interact with the DNA backbone either directly or via hydrogen bond interactions. All these residues are conserved to a high degree in the Prospero/Prox class homeobox proteins. Of course, final confirmation of the HPD-DNA interactions discussed above must await the structure determination of the HPD structure in complex with its DNA binding site (Ryter, 2002).

Therefore, instead of forming an essentially independent unit, the Prospero domain is shown to join together with the homeodomain to form a larger structural unit that has been named the 'homeo-prospero domain'. Model building suggests that this larger structural unit serves in part to align the Prospero domain region on the DNA target. Also, the Prospero domain region is positioned in such a way that it is able to mask a defined nuclear export signal that is within the homeodomain region (Ryter, 2002).

Subcellular localization of the transcription factor Prospero is dynamic. For example, the protein is cytoplasmic in neuroblasts, nuclear in sheath cells, and degraded in newly formed neurons. The carboxy terminus of Prospero, including the homeodomain and Prospero domain, plays roles in regulating these changes. The homeodomain has two distinct subdomains, which exclude proteins from the nucleus, while the intact homeo/Prospero domain masks this effect. One subdomain is an Exportin-dependent nuclear export signal requiring three conserved hydrophobic residues, which models onto helix 1. Another, including helices 2 and 3, requires proteasome activity to degrade nuclear protein. Finally, the Prospero domain is missing in pros(I13) embryos, thus unmasking nuclear exclusion, resulting in constitutively cytoplasmic protein. Multiple processes direct Prospero regulation of cell fate in embryonic nervous system development (Bi, 2003).

date revised: 10 November 2000

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