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Stefan Albert

Publications and source records attributed to Stefan Albert.

13 recordsLinked to original sources
PubMed2006

Evolution of the Yellow/Major Royal Jelly Protein family and the emergence of social behavior in honey bees.

The genomic architecture underlying the evolution of insect social behavior is largely a mystery. Eusociality, defined by overlapping generations, parental brood care, and reproductive division of labor, has most commonly evolved in the Hymenopteran insects, including the honey bee Apis mellifera. In this species, the Major Royal Jelly Protein (MRJP) family is required for all major aspects of eusocial behavior. Here, using data obtained from the A. mellifera genome sequencing project, we demonstrate that the MRJP family is encoded by nine genes arranged in an approximately 60-kb tandem array. Furthermore, the MRJP protein family appears to have evolved from a single progenitor gene that encodes a member of the ancient Yellow protein family. Five genes encoding Yellow-family proteins flank the genomic region containing the genes encoding MRJPs. We describe the molecular evolution of these protein families. We then characterize developmental-stage-specific, sex-specific, and caste-specific expression patterns of the mrjp and yellow genes in the honey bee. We review empirical evidence concerning the functions of Yellow proteins in fruit flies and social ants, in order to shed light on the roles of both Yellow and MRJP proteins in A. mellifera. In total, the available evidence suggests that Yellows and MRJPs are multifunctional proteins with diverse, context-dependent physiological and developmental roles. However, many members of the Yellow/MRJP family act as facilitators of reproductive maturation. Finally, it appears that MRJP protein subfamily evolution from the Yellow protein family may have coincided with the evolution of honey bee eusociality.

Animals↗
PubMed2006

Reversible membrane interaction of BAD requires two C-terminal lipid binding domains in conjunction with 14-3-3 protein binding.

BAD is a Bcl-2 homology domain 3 (BH3)-only proapoptotic member of the Bcl-2 protein family that is regulated by phosphorylation in response to survival factors. Binding of BAD to mitochondria is thought to be exclusively mediated by its BH3 domain. We show here that BAD binds to lipids with high affinities, predominantly to negatively charged phospholipids, such as phosphatidylserine, phosphatidic acid, and cardiolipin, as well as to cholesterol-rich liposomes. Two lipid binding domains (LBD1 and LBD2) with different binding preferences were identified, both located in the C-terminal part of the BAD protein. BAD facilitates membrane translocation of Bcl-XL in a process that requires LBD2. Integrity of LBD1 and LBD2 is also required for proapoptotic activity in vivo. Phosphorylation of BAD does not affect membrane binding but renders BAD susceptible to membrane extraction by 14-3-3 proteins. BAD can be removed efficiently by 14-3-3zeta, -eta, -tau and lesxs efficiently by other 14-3-3 isoforms. The assembled BAD.14-3-3 complex exhibited high affinity for cholesterol-rich liposomes but low affinity for mitochondrial membranes. We conclude that BAD is a membrane-associated protein that has the hallmarks of a receptor rather than a ligand. Lipid binding is essential for the proapoptotic function of BAD in vivo. The data support a model in which BAD shuttles in a phosphorylation-dependent manner between mitochondria and other membranes and where 14-3-3 is a key regulator of this relocation. The dynamic interaction of BAD with membranes is tied to activation and membrane translocation of Bcl-XL.

14-3-3 Proteins↗
PubMed2006

BuCy RAFs drive cells into MEK addiction.

RAF research is booming since the discovery of mutant B-RAF in approximately 8% of human cancer. One reason for the excitement is the availability of RAF-targeted therapies. RAF inhibitors have been developed because RAF functions at a convergence point of signal transduction. Two recent papers by the groups of Rosen and Marais dramatically advance our understanding of RAF oncogenes in human tumors. The results confirm that the mitogenic cascade (RAF-MEK-ERK) is essential for RAF transformation, that RAF kinases work in concert, and that RAF-transformed cells are hooked on MEK, making them sensitive to growth inhibition by kinase inhibitors.

Animals↗
PubMed2005

Organization and potential function of the mrjp3 locus in four honeybee species.

Royal jelly is a nutritious secretion produced by nurse honeybees to provision queens and growing larvae. Major proteins of royal jelly are mutually similar, and they all belong to the MRJP/yellow protein family (pfam03022). The mrjp3 loci in four traditional honeybee species (Apis mellifera, Apis cerana,Apis dorsata, and Apis florea) were sequenced and found to share high sequence and structural similarities. PCR analyses confirmed the presence of an extensive repetitive region, which showed size and sequence polymorphisms in all species. The evolutionary history of mrjp genes and their repetitive regions was reconstructed from their nucleotide sequences. The analyses proved that the repeat region appeared early in the evolution of the mrjp gene family and that the extreme elongation of the repeat is mrjp3 specific. In the MRJPs was documented a correlation between nitrogen content and repeat length. Therefore, it is argued that the repeat occurred due to a selection for an increase in nitrogen storage for a more efficient nutrition of queens and larvae.

Amino Acid Sequence↗
PubMed2005

Two structurally different defensin genes, one of them encoding a novel defensin isoform, are expressed in honeybee Apis mellifera.

Two defensins showing high mutual similarity have previously been characterized in honeybee Apis mellifera: royalisin, a peptide isolated from the royal jelly, and defensin, found in the hemolymph of bacterially infected bees. Here we show that both these peptides are encoded by the same polymorphic gene, which we termed defensin1. Besides this gene, we identified an additional defensin gene coding for a novel honeybee defensin designated defensin2. The pre-pro-peptide sequence of defensin 2 was inferred from its cDNA. Mature defensin 2 peptide shows 55.8% identity with defensin 1. Sequences of genomic loci of the two defensin genes revealed their different structure. Defensin1 possesses an exon-intron structure unique among arthropoda defensin genes. Its second intron splits exactly the common structural module of defensins from a short amidated C-terminal extension found only in hymenopteran defensins. Transcription of defensin genes in some nurse honeybees tissues was studied by RT-PCR. Both defensins are expressed in heads and thoraces. Defensin1 but not defensin2 mRNA was detected in hyphopharyngeal, mandibular and thoracic salivary glands. Immune response elements were identified by computer analysis of the promoter regions of defensin genes. Their different representation in these genes reflects presumably observed tissue-specific expression of defensins.

Amino Acid Sequence↗
PubMed2004

The MRJP/YELLOW protein family of Apis mellifera: identification of new members in the EST library.

Major royal jelly proteins (named MRJP1-5) of honeybee (Apis mellifera), yellow proteins of Drosophila, together with putative proteins found in several bacteria, form a protein family termed the MRJP/yellow family. Members of the family exert diverse physiological functions and amongst eukaryotes appear to be restricted to the order Insecta. MRJPs constitute about 90% of total protein of royal jelly, which is secreted by nurse bees to feed the queen and growing larvae. We looked for mrjp and yellow homologues in a honeybee brain expressed sequence tags (EST) library. In addition to the five mrjp cDNAs previously characterized, we found three additional cDNAs encoding novel MRJPs and importantly, two cDNAs coding for orthologues of Drosophila yellow proteins. One yellow cDNA and all three cDNAs coding for the novel MRJPs were assembled completely, the sequence of the other yellow homologue was partially assembled. The data we present here supports the view that repeated duplications and functional divergence occurred during the evolution of MRJPs in honeybees, with even closely related MRJPs appearing to perform diverse physiological functions. Conversely, yellow protein orthologues appear to be conserved and thus candidates for maintaining the former function(s) of yellow proteins.

Animals↗
PubMed2004

[Cloning and sequence analysis of mrjp1 cDNA from Apis cerana cerana].

A cDNA library was constructed from eight-day-old worker heads of Apis cerana cerana. A probe derived from part of Apis cerana genomic mrjp3 segment was used to screen this library. A total of 120 positive clones of mrjps were screened out from the cDNA library with DIG-probe. The positive clones were amplified and sequenced with T3/T7 primer. 12 sequences were similar to Apis cerana india and Apis mellifera mrjp1 gene by BLAST analysis. The cDNA sequence analysis indicated that the homology was 93.78% between Apis mellifera mrjp1 and Apis cerana mrjp1 while the homology was 99.36% between Apis cerana cerana and Apis cerana india. This result confirmed in molecular level that Apis cerana cerana and Apis cerana india had a common ancestor since the relationship between Apis cerana and Apis mellifera was more far.

Amino Acid Sequence↗
PubMed2003

Dynamic changes in C-Raf phosphorylation and 14-3-3 protein binding in response to growth factor stimulation: differential roles of 14-3-3 protein binding sites.

Phosphorylation events play a crucial role in Raf activation. Phosphorylation of serines 259 and 621 in C-Raf and serines 364 and 728 in B-Raf has been suggested to be critical for association with 14-3-3 proteins. To study the functional consequences of Raf phosphorylations at these positions, we developed and characterized phosphospecific antibodies directed against 14-3-3 binding epitopes: a monoclonal phosphospecific antibody (6B4) directed against pS621 and a polyclonal antibody specific for B-Raf-pS364 epitope. Although 6B4 detected both C- and B-Raf in Western blots, it specifically recognizes the native form of C-Raf but not B-Raf. Contrary to B-Raf, a kinase-dead mutant of C-Raf was found to be only poorly phosphorylated in the Ser-621 position. Moreover, serine 259 to alanine mutation prevented the Ser-621 phosphorylation suggesting an interdependence between these two 14-3-3 binding domains. Direct C-Raf.14-3-3 binding studies with purified proteins combined with competition assays revealed that the 14-3-3 binding domain surrounding pS621 represents the high affinity binding site, whereas the pS259 epitope mediates lower affinity binding. Raf isozymes differ in their 14-3-3 association rates. The time course of endogenous C-Raf activation in mammalian cells by nerve growth factor (NGF) has been examined using both phosphospecific antibodies directed against 14-3-3 binding sites (6B4 and anti-pS259) as well as phosphospecific antibodies directed against the activation domain (anti-pS338 and anti-pY340/pY341). Time course of Ser-621 phosphorylation, in contrast to Ser-259 phosphorylation, exhibited unexpected pattern reaching maximal phosphorylation within 30 s of NGF stimulation. Phosphorylation of tyrosine 340/341 reached maximal levels subsequent to Ser-621 phosphorylation and was coincident with emergence of kinase activity. Taken together, we found substantial differences between C-Raf.14-3-3 binding epitopes pS259 and pS621 and visualized for the first time the sequence of the essential C-Raf phosphorylation events in mammalian cells in response to growth factor stimulation.

14-3-3 Proteins↗
PubMed2003

A role for exocytosis in the spatial regulation of actin organization.

The efficient organization of the actin cytoskeleton is important for many cellular functions. However, how the local actin organization is regulated in a cell is not well understood. By using yeast mutants defective in actin organization and secretion, we demonstrated that exocytosis plays a role in the spatial regulation of actin organization. Our findings suggest that the actin cytoskeleton, exocytosis, and perhaps endocytosis, may depend on each other for efficiency and reinforce each other.

Actins↗
PubMed2003

The GAP activity of Msb3p and Msb4p for the Rab GTPase Sec4p is required for efficient exocytosis and actin organization.

Polarized growth in Saccharomyces cerevisiae is thought to occur by the transport of post-Golgi vesicles along actin cables to the daughter cell, and the subsequent fusion of the vesicles with the plasma membrane. Previously, we have shown that Msb3p and Msb4p genetically interact with Cdc42p and display a GTPase-activating protein (GAP) activity toward a number of Rab GTPases in vitro. We show here that Msb3p and Msb4p regulate exocytosis by functioning as GAPs for Sec4p in vivo. Cells lacking the GAP activity of Msb3p and Msb4p displayed secretory defects, including the accumulation of vesicles of 80-100 nm in diameter. Interestingly, the GAP activity of Msb3p and Msb4p was also required for efficient polarization of the actin patches and for the suppression of the actin-organization defects in cdc42 mutants. Using a strain defective in polarized secretion and actin-patch organization, we showed that a change in actin-patch organization could be a consequence of the fusion of mistargeted vesicles with the plasma membrane.

Actins↗
PubMed2002

Significance of GTP hydrolysis in Ypt1p-regulated endoplasmic reticulum to Golgi transport revealed by the analysis of two novel Ypt1-GAPs.

We here report on the identification and detailed biochemical characterization of two novel GTPase-activating proteins, Gyp5p and Gyp8p, whose efficient substrate is Ypt1p, a Ypt/Rab-GTPase essential for endoplasmic reticulum-to-Golgi trafficking in yeast. Gyp5p accelerated the intrinsic GTPase activity of Ypt1p 4.2 x 10(4)-fold and, surprisingly, the 40-fold reduced GTP hydrolysis rate of Ypt1(Q67L)p 1.5 x 10(4)-fold. At steady state, the two newly discovered GTPase-activating proteins (GAPs) as well as the previously described Gyp1p, which also uses Ypt1p as the preferred substrate, display different subcellular localization. To add to an understanding of the significance of Ypt1p-bound GTP hydrolysis in vivo, yeast strains expressing the GTPase-deficient Ypt1(Q67L)p and having different Ypt1-GAP genes deleted were created. Depending on the genetic background, different mutants exhibited growth defects at low temperature and, already at permissive temperature, various morphological alterations resembling autophagy. Transport of proteins was not significantly impaired. Growth defects of Ypt1(Q67L)-expressing cells could be suppressed on high expression of all three Ypt1-GAPs. We propose that permanently active Ypt1p leads to increased vesicle fusion, which might induce previously unnoticed autophagic degradation of exaggerated membrane-enclosed structures. The data indicate that hydrolysis of Ypt1p-bound GTP is a prerequisite for a balanced vesicle flow between endoplasmic reticulum and Golgi compartments.

Amino Acid Sequence↗
PubMed2002

Interaction of yeast Rab geranylgeranyl transferase with its protein and lipid substrates.

Small GTPases from the Rab/Ypt family regulate events of vesicular traffic in eukaryotic cells. For their activity, Rab proteins require a posttranslational modification that is conferred by Rab geranylgeranyltransferase (RabGGTase), which attaches geranylgeranyl moieties onto two cysteines of their C terminus. RabGGTase is present in both lower and higher eukaryotes in the form of heterodimers composed of alpha and beta subunits. However, the alpha subunits of RabGGTases from lower eukaryotes, including Saccharomyces cerevisiae (yRabGGTase), are half the size of the corresponding subunit of the mammalian enzyme. This difference is due to the presence of additional immunoglobulin (Ig)-like and leucine rich (LRR) domains in the mammalian transferase. To understand the possible evolutionary implications and functional consequences of structural differences between RabGGTases of higher and lower eukaryotes, we have investigated the interactions of yeast RabGGTase with its lipid and protein substrate. We have demonstrated that geranylgeranyl pyrophosphate binds to the enzyme with an affinity of ca. 40 nM, while binding of farnesyl pyrophosphate is much weaker, with a K(d) value of ca. 750 nM. This finding suggests that despite the structural difference, yRabGGTase selects its lipid substrate in a fashion similar to mammalian RabGGTase. However, unlike the mammalian enzyme, yRabGGTase binds prenylated and unprenylated Ypt1p:Mrs6p complexes with similar affinities (K(d) ca. 200 nM). Moreover, in contrast to the mammalian enzyme, phosphoisoprenoids do not influence the affinity of Mrs6p for yRabGGTase. Using an in vitro prenylation assay, we have demonstrated that yRabGGTase can prenylate Rab proteins in complex with mammalian REP-1, thus indicating that neither the LRR nor the Ig-like domains, nor the recently discovered alternative pathway of catalytic complex assembly, are essential for the catalytic activity of RabGGTase. Despite the ability to function in concert with yRabGGTase in vitro, expression of mammalian REP-1 could not complement deletion of MRS6 gene in S. cerevisiae in vivo. The implications of these findings are discussed.

Adaptor Proteins, Signal Transducing↗