Identification of a 521-kilodalton protein (Gli521) involved in force generation or force transmission for Mycoplasma mobile gliding

Several mycoplasma species are known to glide on solid surfaces such as glass in the direction of the membrane protrusion, but the mechanism underlying this movement is unknown. To identify a novel protein involved in gliding, we raised monoclonal antibodies against a detergent-insoluble protein fraction of Mycoplasma mobile, the fastest glider, and screened the antibodies for inhibitory effects on gliding. Five monoclonal antibodies stopped the movement of gliding mycoplasmas, keeping them on the glass surface, and all of them recognized a large protein in immunoblotting. This protein, named Gli521, is composed of 4,738 amino acids, has a predicted molecular mass of 520,559 Da, and is coded downstream of a gene for another gliding protein, Gli349, which is known to be responsible for glass binding during gliding. Edman degradation analysis indicated that the N-terminal region is processed at the peptide bond between the amino acid residues at positions 43 and 44. Analysis of gliding mutants isolated previously revealed that the Gli521 protein is missing in a nonbinding mutant, m9, where the gli521 gene is truncated by a nonsense mutation at the codon for the amino acid at position 1170. Immunofluorescence and immunoelectron microscopy indicated that Gli521 localizes all around the base of the membrane protrusion, at the "neck," as previously observed for Gli349. Analysis of the inhibitory effects of the anti-Gli521 antibody on gliding motility revealed that this protein is responsible for force generation or force transmission, a role distinct from that of Gli349, and also suggested conformational changes of Gli349 and Gli521 during gliding.     

Molecular shape and binding force of Mycoplasma mobile’s leg protein Gli349 revealed by an AFM study

Recent studies of the gliding bacteria Mycoplasma mobile have identified a family of proteins called the Gli family which was considered to be involved in this novel and yet fairly unknown motility system. The 349 kDa protein called Gli349 was successfully isolated and purified from the bacteria, and electron microscopy imaging and antibody experiments led to the hypothesis that it acts as the “leg” of M. mobile, responsible for attachment to the substrate as well as for gliding motility. However, more precise evidence of the molecular shape and function of this protein was required to asses this theory any further. In this study, an atomic force microscope (AFM) was used both as an imaging and a force measurement device to provide new information about Gli349 and its role in gliding motility. AFM images of the protein were obtained revealing a complex structure with both rigid and flexible parts, consistent with previous electron micrographs of the protein. Single-molecular force spectroscopy experiments were also performed, revealing that Gli349 is able to specifically bind to sialyllactose molecules and withstand unbinding forces around 70 pN. These findings strongly support the idea that Gli349 is the “leg” protein of M. mobile, responsible for binding and also most probably force generation during gliding motility.     

Morphology of isolated Gli349, a leg protein responsible for Mycoplasma mobile gliding via glass binding, revealed by rotary shadowing electron microscopy

Several species of mycoplasmas rely on an unknown mechanism to glide across solid surfaces in the direction of a membrane protrusion at the cell pole. Our recent studies on the fastest species, Mycoplasma mobile, suggested that a 349-kDa protein, Gli349, localized at the base of the membrane protrusion called the neck, forms legs that stick out from the neck and propel the cell by repeatedly binding to and releasing from a solid surface, based on the energy of ATP hydrolysis. Here, the Gli349 protein was isolated from mycoplasma cells and its structure was analyzed. Gel filtration analysis showed that the isolated Gli349 protein is monomeric. Rotary shadowing electron microscopy revealed that the molecular structure resembles the symbol for an eighth note in music. It contains an oval foot 14 nm long in axis. From this foot extend three rods in tandem of 43, 20, and 20 nm, in that order. The hinge connecting the first and second rods is flexible, while the next hinge has a distinct preference in its angle, near 90 degrees. Molecular images revealed that a monoclonal antibody that can bind to the position at one-third of the total peptide length from the N terminus bound to a position two-thirds from the foot end, suggesting that the foot corresponds to the C-terminal region. The amino acid sequence was assigned to the molecular image, and the topology of the molecule in the gliding machinery is discussed.     

Spike Structure at the Interface between Gliding Mycoplasma mobile Cells and Glass Surfaces Visualized by Rapid-Freeze-and-Fracture Electron Microscopy

Mycoplasma mobile is a flask-shaped bacteria that binds to a substrate and glides towards its tapered end, the so-called "head-like protrusion," by an unknown mechanism. To search for cellular structures underlying this motility, the cell-substrate interface of actively gliding cells was visualized by rapid-freeze-and-freeze-fracture rotary-shadow electron microscopy. Novel structures, called "spikes," were observed to protrude from the cell membrane and attach to the glass surface at their distal end. The spikes were on average 50 nm in length and 4 nm in diameter, most abundant around the head, and not observed in a nonbinding mutant. The spikes may be involved in the mechanism of binding, gliding, or both.     

Movement on the cell surface of the gliding bacterium,Mycoplasma mobile,is limited to its head-like structure

Mycoplasma mobile cells glide on solid surfaces such as glass with a fast and continuous motion in the direction of the membrane protrusion (head-like structure) at one cell pole. To examine its cell-surface movement, a latex bead was attached to a cell and behavior in gliding was monitored. The bead was carried without movement relative to the cell body, suggesting that the cell does not roll around the cell axis and the surface movement is limited to a small area. A small percentage of cells showed an elongated head-like structure in an old batch culture. The head-like structure moved forward, sometimes leaving the cell body in one position, resulting in a stretching of this head-like structure. These results indicate that the head-like structure drags the cell body, leading us to conclude that the force for gliding is generated at the head-like structure.     

Gliding motility of Mycoplasma mobile can occur by repeated binding to N-acetylneuraminyllactose (sialyllactose) fixed on solid surfaces

Mycoplasma mobile relies on an unknown mechanism to glide across solid surfaces including glass, animal cells, and plastics. To identify the direct binding target, we examined the factors that affect the binding of Mycoplasma pneumoniae to solid surfaces and concluded that N-acetylneuraminyllactose (sialyllactose) attached to a protein can mediate glass binding on the basis of the following four lines of evidence: (i) glass binding was inhibited by N-acetylneuraminidase, (ii) glass binding was inhibited by N-acetylneuraminyllactose in a structure-dependent manner, (iii) binding occurred on glass pretreated with bovine serum albumin attached to N-acetylneuraminyllactose, and (iv) gliding speed depended on the density of N-acetylneuraminyllactose on glass.     

The Severe Autosomal Dominant Retinitis Pigmentosa Rhodopsin Mutant Ter349Glu Mislocalizes and Induces Rapid Rod Cell Death

Mutations in the rhodopsin gene cause approximately one-tenth of retinitis pigmentosa cases worldwide, and most result in endoplasmic reticulum retention and apoptosis. Other rhodopsin mutations cause receptor mislocalization, diminished/constitutive activity, or faulty protein-protein interactions. The purpose of this study was to test for mechanisms by which the autosomal dominant rhodopsin mutation Ter349Glu causes an early, rapid retinal degeneration in patients. The mutation adds an additional 51 amino acids to the C terminus of the protein. Folding and ligand interaction of Ter349Glu rhodopsin were tested by ultraviolet-visible (UV-visible) spectrophotometry. The ability of the mutant to initiate phototransduction was tested using a radioactive filter binding assay. Photoreceptor localization was assessed both in vitro and in vivo utilizing fluorescent immunochemistry on transfected cells, transgenic Xenopus laevis, and knock-in mice. Photoreceptor ultrastructure was observed by transmission electron microscopy. Spectrally, Ter349Glu rhodopsin behaves similarly to wild-type rhodopsin, absorbing maximally at 500 nm. The mutant protein also displays in vitro G protein activation similar to that of WT. In cultured cells, mislocalization was observed at high expression levels whereas ciliary localization occurred at low expression levels. Similarly, transgenic X. laevis expressing Ter349Glu rhodopsin exhibited partial mislocalization. Analysis of the Ter349Glu rhodopsin knock-in mouse showed a rapid, early onset degeneration in homozygotes with a loss of proper rod outer segment development and improper disc formation. Together, the data show that both mislocalization and rod outer segment morphogenesis are likely associated with the human phenotype.     

Identification of a novel nucleoside triphosphatase from Mycoplasma mobile: a prime candidate motor for gliding motility

A protein with a molecular mass of 42 kDa (P42) from Mycoplasma mobile, one of several mycoplasmas that exhibit gliding motility, was shown to be a novel NTPase (nucleoside triphosphatase). Although the P42 protein lacks a common ATP-binding sequence motif (Walker A), the recombinant proteins expressed in Escherichia coli certainly hydrolysed some nucleoside triphosphates, including ATP. The results of photoaffinity labelling by an ATP analogue supported that the P42 protein contains a specific binding site for ATP (or another nucleoside triphosphate). In the M. mobile genome, the P42 gene is located downstream of gli123, gli349 and gli521 genes, and they have been reported to be polycis-tronically transcribed. As the huge proteins encoded by gli123, gli349 and gli521 play a role in gliding motility of M. mobile, P42 might also have some kind of function in the gliding motility. The gliding motility of M. mobile is driven directly by ATP hydrolysis, but the key ATPase has not been identified. Our results showed that, among these four proteins, only P42 exhibited ATPase activity. Biochemical characteristics--optimal conditions for activity, substrate specificities, and inhibiting effects by ATP analogues--of the recombinant P42 proteins were very similar to those of a putative ATPase speculated from a previous analysis with a gliding 'ghost' whose cell membrane was permeabilized by Triton X-100. These results support the hypothesis that the P42 protein is the key ATPase in the gliding motility of M. mobile.     

Agonist-independent internalization and activity of a C-terminally truncated somatostatin receptor subtype 2 (delta349)

The rat somatostatin receptor subtype 2 (SSTR2) is rapidly internalized and phosphorylated in the presence of somatostatin 14 (SST14). Several C-terminal deletion constructs of SSTR2 have been investigated for their ability to undergo agonist-dependent internalization by using biochemical ligand binding assays and confocal microscopic analysis. Whereas mutant receptors lacking either 10 (delta359), 30 (delta339), or 44 (delta325) amino acid residues at the C terminus required SST14 for internalization, a construct lacking the last 20 amino acids (delta349) was detected mostly intracellularly and independently of the presence of the agonist. When internalization was blocked by sucrose, the delta349 receptor remained at the cell surface, strongly indicating that this mutant is internalized in an agonist-independent fashion. An increased affinity for agonists as measured in membrane binding assays and a reduced level of forskolin-stimulated cyclic AMP accumulation in human embryonic kidney cells expressing delta349 are properties that are characteristic of agonist-independent receptor activity. Delta349 is not phosphorylated detectably in the absence of agonist, demonstrating that phosphorylation per se is not a prerequisite for internalization of SSTR2. This observation is in line with data obtained for the delta325 mutant, which was internalized in an agonist-dependent manner, but not phosphorylated in either the presence or absence of SST14. We conclude that truncation of the SSTR2 C terminus at position 349 leads to agonist-independent, constitutive activity and internalization.     

Aspartic acid 349 in the fourth epidermal growth factor-like structure of human thrombomodulin plays a role in its Ca(2+)-mediated binding to protein C.

The last three consecutive epidermal growth factor (EGF)-like structures of human thrombomodulin constitute the functional domain for protein C-activating cofactor activity and anticoagulant activity. Using site-directed deletion mutagenesis, we found that amino acid Asp349 of TME456, a recombinantly produced protein consisting of EGF-like structures 4, 5, and 6, is essential for retaining full protein C-activating cofactor activity. To investigate the role of Asp349 in the protein C-activating cofactor activity of human thrombomodulin, we have constructed two mutants of TMD123, a recombinantly produced protein consisting of domains D1, D2, and D3 of thrombomodulin, using site-directed point mutagenesis of the thrombomodulin coding sequence. In mutant TMD123A, the Asp349 codon was replaced with an Ala codon and in mutant TMD123E, the Asp349 codon was replaced with a Glu codon. The partially purified mutant proteins were assayed for their protein C-activating cofactor activity at various Ca2+ concentrations. TMD123 and TMD123E protein showed similar high levels of cofactor activity and similar patterns of Ca2+ dependence, while TMD123A had lower cofactor activity and did not show any Ca2+ dependence. We concluded that Asp349 in the fourth EGF-like structure of human thrombomodulin plays a role in its Ca(2+)-mediated binding to protein C.     

Mutant analysis reveals a specific requirement for protein P30 in Mycoplasma pneumoniae gliding motility

The cell-wall-less prokaryote Mycoplasma pneumoniae, long considered among the smallest and simplest cells capable of self-replication, has a distinct cellular polarity characterized by the presence of a differentiated terminal organelle which functions in adherence to human respiratory epithelium, gliding motility, and cell division. Characterization of hemadsorption (HA)-negative mutants has resulted in identification of several terminal organelle proteins, including P30, the loss of which results in developmental defects and decreased adherence to host cells, but their impact on M. pneumoniae gliding has not been investigated. Here we examined the contribution of P30 to gliding motility on the basis of satellite growth and cell gliding velocity and frequency. M. pneumoniae HA mutant II-3 lacking P30 was nonmotile, but HA mutant II-7 producing a truncated P30 was motile, albeit at a velocity 50-fold less than that of the wild type. HA-positive revertant II-3R producing an altered P30 was unexpectedly not fully wild type with respect to gliding. Complementation of mutant II-3 with recombinant wild-type and mutant alleles confirmed the correlation between gliding defect and loss or alteration in P30. Surprisingly, fusion of yellow fluorescent protein to the C terminus of P30 had little impact on cell gliding velocity and significantly enhanced HA. Finally, while quantitative examination of HA revealed clear distinctions among these mutant strains, gliding defects did not correlate strictly with the HA phenotype, and all strains attached to glass at wild-type levels. Taken together, these findings suggest a role for P30 in gliding motility that is distinct from its requirement in adherence.     

Arginine-349 and aspartate-373 of the Na(+)/dicarboxylate cotransporter are conformationally sensitive residues.

The conserved residues, Arg-349 and Asp-373, of the renal Na(+)/dicarboxylate cotransporter (NaDC-1) have been shown in our previous studies to affect substrate affinity and cation binding. In this study, amino acids surrounding Arg-349 and Asp-373 were individually mutated to cysteines and their sensitivity to methanethiosulfonate reagents (MTS) was tested. Only three of the 21 mutants were sensitive to MTS reagents: R349C, S372C, and D373C. The R349C mutant had reduced activity which was restored by chemical modification with MTSEA. The effect of MTSEA was only observed in the presence of sodium, indicating that Arg-349 is conformationally accessible. The succinate transport activity of the S372C mutant was stimulated by both MTSEA and MTSET. The D373C mutant was very sensitive to inhibition by MTSET (K(i) = 0.5 microM) in sodium buffer. The inhibition of D373C by MTSET was prevented by substrate, suggesting that the substrate-induced conformational change occludes the residue. We conclude that the accessibility of Arg-349 and Asp-373 is likely to change with the conformational states of the transport cycle.     

Triskelion Structure of the Gli521 Protein,Involved in the Gliding Mechanism of Mycoplasma mobile

Mycoplasma mobile binds to solid surfaces and glides smoothly and continuously by a unique mechanism. A huge protein, Gli521 (521 kDa), is involved in the gliding machinery, and it is localized in the cell neck, the base of the membrane protrusion. This protein is thought to have the role of force transmission. In this study, the Gli521 protein was purified from M. mobile cells, and its molecular shape was studied. Gel filtration analysis showed that the isolated Gli521 protein forms mainly a monomer in Tween 80-containing buffer and oligomers in Triton X-100-containing buffer. Rotary shadowing electron microscopy showed that the Gli521 monomer consisted of three parts: an oval, a rod, and a hook. The oval was 15 nm long by 11 nm wide, and the filamentous part composed of the rod and the hook was 106 nm long and 3 nm in diameter. The Gli521 molecules form a trimer, producing a “triskelion” reminiscent of eukaryotic clathrin, through association at the hook end. Image averaging of the central part of the triskelion suggested that there are stable and rigid structures. The binding site of a previously isolated monoclonal antibody on Gli521 images showed that the hook end and oval correspond to the C- and N-terminal regions, respectively. Partial digestion of Gli521 showed that the molecule could be divided into three domains, which we assigned to the oval, rod, and hook of the molecular image. The Gli521 molecule''s role in the gliding mechanism is discussed.Mycoplasmas are commensal and occasionally parasitic bacteria with small genomes that lack a peptidoglycan layer (31). Several mycoplasma species form membrane protrusions, such as the headlike structure in Mycoplasma mobile and the attachment organelle in Mycoplasma pneumoniae (15, 19, 21, 22, 25, 33, 34, 36). On solid surfaces, these species exhibit gliding motility in the direction of the protrusion; this motility is believed to be involved in the pathogenicity of mycoplasmas (12, 13, 16, 20, 21). Interestingly, mycoplasmas have no surface flagella or pili, and their genomes contain no genes related to other known bacterial motility systems. In addition, no homologs of motor proteins that are common in eukaryotic motility have been found (11).M. mobile, which was isolated from the gills of a freshwater fish in the early 1980s, is a fast gliding mycoplasma (14). It glides smoothly and continuously on glass at an average speed of 2.0 to 4.5 μm/s, or three to seven times the length of the cell per second, exerting a force of up to 27 pN (8, 9, 24, 25, 32). Previously, we identified huge proteins involved in this gliding mechanism that are localized at the so-called cell neck, the base of the membrane protrusion (17, 26, 30, 35, 37, 39); we also visualized the putative machinery and the binding protein (1, 18, 23) and identified both the direct energy source used and the direct binding target (10, 27, 38). The force generated by the gliding machinery may be supported from inside the cell by a cytoskeletal “jellyfish” structure (28, 29). On the basis of these results, we proposed a working model, called the centipede or power stroke model, where cells are propelled by “legs” composed of Gli349 that repeatedly catch and release sialic acids fixed on the glass surface (5, 19, 21). These legs are driven by the force exerted by P42 through Gli521 molecules, which is supported by the jellyfish structure, based on energy from ATP hydrolysis.The Gli521 protein, which has an unusually high molecular mass (521 kDa), is suggested to have the role of force transmission, because a monoclonal antibody against this protein stops gliding, keeping the cells on a solid surface (35). About 450 molecules are estimated to be clustered in the gliding machinery with other component proteins, although their alignment has not been clarified (35, 37, 39). In this study, we isolated the Gli521 protein and studied its molecular shape using electron microscopy (EM) and biochemical analyses in order to understand the gliding mechanism.     

Mutations in Flavobacterium johnsoniae sprE result in defects in gliding motility and protein secretion

Cells of the gliding bacterium Flavobacterium johnsoniae move rapidly over surfaces. Transposon mutagenesis was used to identify sprE, which is involved in gliding. Mutations in sprE resulted in the formation of nonspreading colonies on agar. sprE mutant cells in wet mounts were almost completely deficient in attachment to and movement on glass, but a small percentage of cells exhibited slight movements, indicating that the motility machinery was not completely disrupted. SprE is a predicted lipoprotein with a tetratricopeptide repeat domain. SprE is similar in sequence to Porphyromonas gingivalis PorW, which is required for secretion of gingipain protease virulence factors. Disruption of F. johnsoniae sprE resulted in decreased extracellular chitinase activity and decreased secretion of the cell surface motility protein SprB. Reduced secretion of cell surface components of the gliding machinery, such as SprB, may account for the defects in gliding. Orthologs of sprE are found in many gliding and nongliding members of the phylum Bacteroidetes, suggesting that similar protein secretion systems are common among members of this large and diverse group of bacteria.     

禽流感病毒血凝素HA的可变性解析

以重组的蒙古鸭H5N2禽流感病毒A/Duck/Mongolia/54/01的血凝素HA蛋白的cDNA为模板,进行PCR随机突变,表达只有单个氨基酸突变的H5HA基因共计38个.根据红细胞吸附反应,分析这些突变HA的功能,仍然具有红细胞吸附活性的单个氨基酸突变的HA约占89%,说明H5HA单个氨基酸突变的容许率是相当高的.HA1区突变数目大约是HA2区的两倍.对失去红细胞吸附功能和某些仍然拥有红细胞吸附功能的HA及单个氨基酸突变的位置与结构的关系进行探讨.有两个位点氨基酸突变了两次,但都不影响红细胞吸附功能,对红细胞吸附功能的影响,似乎主要由位置决定,而不是取决于取代的氨基酸的种类.位点179位和122位的突变是不允许的;位点179位于H5N1的受体结合区域RBD内,122位位于A抗原决定簇区附近,推测在H5HA三维结构上,这两个位点位于HA分子的内部,维持着H5HA的结构.HA1Cys位点4和HA2Cys位点148的突变是不允许的.这两个Cys正好形成HA1和HA2连接的桥梁,对维持H5HA结构也是相当重要的.本实验中HA先后失去了三个糖基化位点,但并不影响吸附红细胞的功能.总之,通过实验分析以研究某些氨基酸改变的效果,寻找关键位点是否突变,可以作为评估H5N1野毒株大流行潜力的分子标志.     

Involvement of P1 adhesin in gliding motility of Mycoplasma pneumoniae as revealed by the inhibitory effects of antibody under optimized gliding conditions

To examine the participation of P1 adhesin in gliding of Mycoplasma pneumoniae, we examined the effects of an anti-P1 monoclonal antibody on individual gliding mycoplasmas. The antibody reduced the gliding speed and removed the gliding cells from the glass over time in a concentration-dependent manner but had only a slight effect on nongliding cells, suggesting that the conformational changes of P1 adhesin and its displacement are involved in the gliding mechanism.     

Characterization of point mutations in patients with pyruvate dehydrogenase deficiency: role of methionine-181, proline-188, and arginine-349 in the alpha subunit.

Human pyruvate dehydrogenase (E1), a heterotetramer (alpha2beta2), is the first component of the pyruvate dehydrogenase complex (PDC). E1 catalyzes the thiamin pyrophosphate (TPP)-dependent decarboxylation of pyruvate and the reductive acetylation of the dihydrolipoamide acetyltransferase component. Site-directed mutagenesis was employed to recreate three point mutations in the alpha subunit identified in E1-deficient patients, M181V, R349H, and P188L (P188A mutant E1 was used because of the very low level of expression of P188L), to investigate the functional roles of these three amino acid residues. P188A mutant E1 was much less thermostable than the wild-type E1. The kcats of M181V and P188A mutant E1s determined in the PDC reaction were 38 and 24% of that of the wild-type enzyme, respectively. The apparent Km for TPP for M181V increased significantly (approx 250-fold when determined in the PDC assay), while the apparent Km for pyruvate increased by only about 3-fold. In contrast, P188A had similar Kms for the coenzyme and the substrate as the wild-type. Km values for R349H were not determined due to the extremely low activity of this mutant (1.2% of the wild-type E1-specific activity measured in the PDC assay). Wild-type E1 displayed a lag phase in the progress curve of the PDC reaction measured in the presence of low TPP concentrations (below 1 microM) only. All mutants had a lag phase that was not eliminated even at very high TPP concentrations, suggesting modifications in the conformation of the active site. Kinetic analysis indicated thiamin 2-thiothiazolone pyrophosphate (ThTTPP) to be an intermediate analog for wild-type human E1. M181V required a higher concentration of ThTTPP for inactivation than the wild-type and P188A E1s. The results of circular dichroism spectropolarimetry in the far UV region indicated that there were no major changes in the secondary structure of M181V, P188A, and R349H E1s. These mutant enzymes exhibited negative dichroic spectra at about 330 nm only in the presence of high TPP concentrations. This study suggests that arginine-349 is critical for E1's activity, methionine-181 is involved in the binding of TPP, and proline-188 is necessary for structural integrity of E1.     

Aurora-A kinase Ser349 phosphorylation is required during Xenopus laevis oocyte maturation

Xenopus laevis Aurora-A is phosphorylated in vivo onto three amino acids: Ser53, Thr295 and Ser349. The activation of the kinase depends on its autophosphorylation on Thr295 within the T-loop. The phosphorylation of Ser53 by still unknown kinase(s) prevents its degradation. The present work focused on the regulation of Aurora-A function via Ser349 phosphorylation. Mutagenesis of Ser349 to alanine (S349A) had few impact in vitro on the capability of the kinase to autophosphorylate as well as on its activity. These data in addition to in gel kinase assays and site-specific proteolytic digestion experiments prove that Ser349 is clearly neither a primary autophosphorylation site, nor an autophosphorylation site depending on the priming phosphorylation of Thr295. Using specific antibodies, we also show that the phosphorylation of Aurora-A Ser349 is a physiological event during Xenopus oocyte maturation triggered by progesterone. A peak of phosphorylation paralleled the decrease of Aurora activity observed between meiosis I and II. In response to progesterone, X. laevis stage VI oocytes microinjected with the Aurora-A S349A mutant proceeded normally to germinal vesicle breakdown (GVBD), but degenerated rapidly soon after. Since phosphorylation of Ser349 is responsible for a decrease in kinase activity, our results suggest that a down-regulation of Aurora-A activity involving Ser349 phosphorylation is required in the process of maturation.