Roles of an N-terminal coiled-coil-containing domain in the localization and function of Bem3, a Rho GTPase-activating protein in budding yeast.

GTPase-activating proteins (GAPs) play critical roles in the spatial and temporal control of small GTPases. The budding yeast Bem3 is a GAP for Cdc42, a Rho GTPase crucial for actin and septin organization. Bem3 localizes to the sites of polarized growth. However, the amino acid sequence determinants mediating recruitment of Bem3 to its physiological sites of action and those important for Bem3 function are not clear. Here, we show that Bem3's localization is guided by two distinct targeting regions-the PX-PH-domain-containing TD1 and the coiled-coil-containing TD2. TD2 localization is largely mediated by its interaction with the polarisome component Epo1 via heterotypic coiled-coil interaction. This finding reveals a novel role for the polarisome in linking Bem3 to its functional target, Cdc42. We also show that the coiled-coil domain of Bem3 interacts homotypically and this interaction is important for the regulation of Cdc42 by Bem3. Moreover, we show that overexpression of a longer version of the TD2 domain disrupts septin-ring assembly in a RhoGAP-independent manner, suggesting that TD2 may be capable of interacting with proteins implicated in septin-ring assembly. Furthermore, we show that the longer version of TD2 interacts with Kss1, a MAPK involved in filamentous growth. Kss1 is reported to localize mainly in the nucleus. We find that Kss1 also localizes to the sites of polarized growth and Bem3 interacts with Kss1 at the septin-ring assembly site. Our study provides new insights in Bem3's localization and function.

Kin2, the Budding Yeast Ortholog of Animal MARK/PAR-1 Kinases, Localizes to the Sites of Polarized Growth and May Regulate Septin Organization and the Cell Wall.

MARK/PAR-1 protein kinases play important roles in cell polarization in animals. Kin1 and Kin2 are a pair of MARK/PAR-1 orthologs in the budding yeast Saccharomyces cerevisiae. They participate in the regulation of secretion and ER stress response. However, neither the subcellular localization of these two kinases nor whether they may have other cellular functions is clear. Here, we show that Kin2 localizes to the sites of polarized growth in addition to localization on the plasma membrane. The localization to polarity sites is mediated by two targeting domains-TD1 and TD2. TD1 locates in the N-terminal region that spans the protein kinase domain whereas TD2 locates in the C-terminal end that covers the KA1 domain. We also show that an excess of Kin2 activity impaired growth, septin organization, and chitin deposition in the cell wall. Both TD1 and TD2 contribute to this function. Moreover, we find that the C-terminal region of Kin2 interacts with Cdc11, a septin subunit, and Pea2, a component of the polarisome that is known to play a role in septin organization. These findings suggest that Kin2 may play a role in the regulation of the septin cytoskeleton and the cell wall. Finally, we show that the C-terminal region of Kin2 interacts with Rho3, a Rho GTPase, whereas the N-terminal region of Kin2 interacts with Bmh1, a 14-3-3 protein. We speculate that Kin2 may be regulated by Bmh1, Rho3, or Pea2 in vivo. Our study provides new insight in the localization, function, and regulation of Kin2.

The GTPase-activating protein Rga1 interacts with Rho3 GTPase and may regulate its function in polarized growth in budding yeast.

In budding yeast, Rga1 negatively regulates the Rho GTPase Cdc42 by acting as a GTPase-activating protein (GAP) for Cdc42. To gain insight into the function and regulation of Rga1, we overexpressed Rga1 and an N-terminally truncated Rga1-C538 (a.a. 538-1007) segment. Overexpression of Rga1-C538 but not full-length Rga1 severely impaired growth and cell morphology in wild-type cells. We show that Rga1 is phosphorylated during the cell cycle. The lack of phenotype for full-length Rga1 upon overexpression may result from a negative regulation by G1-specific Pho85, a cyclin-dependent kinase (CDK). From a high-copy suppressor screen, we isolated RHO3, SEC9, SEC1, SSO1, SSO2, and SRO7, genes involved in exocytosis, as suppressors of the growth defect caused by Rga1-C538 overexpression. Moreover, we detected that Rga1 interacts with Rho3 in two-hybrid and bimolecular fluorescence complementation (BiFC) assays. Rga1 preferentially interacts with the GTP-bound form of Rho3 and the interaction requires the GAP domain and additional sequence upstream of the GAP domain. Our data suggest that the interaction of Rga1 with Rho3 may regulate Rho3's function in polarized bud growth.

[Application of fluorescence in situ hybridization to prenatal diagnosis of aneuploidy in 110 uncultured amniotic fluid samples].

OBJECTIVE: To optimize the prenatal diagnosis platform by using domestically made fluorescence in situ hybridization(FISH) kit and to explore the clinical application of FISH to rapid prenatal diagnosis of a wide range of chromosomal abnormalities. METHODS: Amniotic fluid samples from 110 pregnant women were studied with the rapid prenatal diagnosis method of FISH and the conventional cell culture method of karyotyping, the results from both methods were compared. RESULTS: Four cases of trisomy 21, 1 case of trisomy 18, 58 cases of 46, XX, and 47 cases of 46, XY were detected by FISH in the 110 amniotic fluid samples. It is concordant with the results from conventional karyotype analysis. The concordance rate is 100%. CONCLUSION: Domestically made FISH kit can be used to rapidly and accurately detect the most common chromosome aneuploidies by using less sample volume while the price is relatively low. FISH can be a reliable and rapid prenatal diagnostic tool as an adjunct to classical cytogenetic study. It can be used for rapid and accurate prenatal diagnosis of women with high risk of maternal serum screening.

[Application of spectral karyotyping in diagnosis and prenatal diagnosis of the marker chromosome].

OBJECTIVE: To determine the value of spectral karyotyping(SKY) in identification of the marker chromosome. METHODS: Selected six cases that could not be identified in clinic were studied, using samples of peripheral blood from four cases, and samples of amonic fluid and fetal cord blood for prenatal diagnosis in two cases were investigated. All cases were analyzed with the routine SKY method, and the results with the SKY View software. The SKY results were identified by using fluorescence in situ hybridization (FISH). And C-banding technique was used to help diagnose the heterochromatin. RESULTS: SKY was successfully performed on all of 6 cases. The origin of all marker chromosomes was identified by SKY. Except case No. 4, the others were confirmed by FISH. It helped determine the pregnancy outcome in two cases of prenatal diagnosis: one case of genetic marker chromosome continued the pregnancy, and another case of de novo marker chromosome was terminated of the pregnancy. CONCLUSION: SKY may be a valuable tool to diagnose the marker chromosome with rapidness,direct-viewing and sensitiveness. It can be used to assess the prognosis and the pregnancy outcome.

[Application of spectral karyotyping in diagnosis of complex chromosome aberration].

OBJECTIVE: To determine the value of spectral karyotyping (SKY) to identify the complex chromosome aberration. METHODS: Four cases were selected that can not be identified by standard cytogenetic techniques. The chromosome specimens were detected by the routine SKY method, and the results were analyzed by the SKY View software. RESULTS: By using SKY a case of complex chromosome rearrangements and two cases of chromosome duplication were identified. However it could not identify the chromosome inversion and the breakpoint of chromosome aberration. CONCLUSION: SKY may be a valuable tool in identification of complex chromosome translocation, rearrangement, minute aberration and unknown derivative chromosomes. Though SKY can not replace the standard cytogenetic techniques, but it will be the benefit supplementary.

[HLA-B gene polymorphism detected by high-resolution sequence-based typing in Guangdong Han populations].

OBJECTIVE: To investigate the genetic polymorphism of HLA-B locus in Guangdong Han population and compare the characteristic of the allele frequency distribution with that in other populations. METHODS: A total of 562 cord blood samples from Guangzhou Cord Blood Bank were analyzed by sequence-based typing. Then the sequences encompassing exons 2, 3, and 4 for HLA-B gene were analyzed by direct sequencing of PCR products. The allele frequency distribution of HLA-B in this population was compared with that in other populations. RESULTS: A total of 59 different HLA-B alleles were detected, and among them were 6 HLA-B alleles with frequencies higher than 5%: HLA-B*4601 (14.5%), HLA-B*400101 (14.4%), HLA-B*1502 (11.5%), HLA-B*1301 (8.6%), HLA-B*5801 (8.1%) and HLA-B*380201 (6.4%); the total frequency of these six alleles was 63.5%. At the same time, there were 30 kinds of HLA-B allele with frequencies lower than 0.5%; the total frequency of these alleles was 4.9%. Maximum variation at HLA-B was seen in the HLA-B*15 allele family (nine alleles). Comparison of the HLA-B frequencies in different populations showed a close relationship of Guandong Han population with the Chinese populations from Hong Kong and Singapore, respectively. CONCLUSION: The results have shown the characteristic of HLA-B distribution and provided more accurate genotypic data that may serve as normal reference value for the Han population in Guangdong, China.