Widespread use of unconventional targeting signals in mitochondrial ribosome proteins

Mitochondrial ribosomes are complex molecular machines indispensable for respiration. Their assembly involves the import of several dozens of mitochondrial ribosomal proteins (MRPs), encoded in the nuclear genome, into the mitochondrial matrix. Proteomic and structural data as well as computational predictions indicate that up to 25% of yeast MRPs do not have a conventional N‐terminal mitochondrial targeting signal (MTS). We experimentally characterized a set of 15 yeast MRPs in vivo and found that five use internal MTSs. Further analysis of a conserved model MRP, Mrp17/bS6m, revealed the identity of the internal targeting signal. Similar to conventional MTS‐containing proteins, the internal sequence mediates binding to TOM complexes. The entire sequence of Mrp17 contains positive charges mediating translocation. The fact that these sequence properties could not be reliably predicted by standard methods shows that mitochondrial protein targeting is more versatile than expected. We hypothesize that structural constraints imposed by ribosome assembly interfaces may have disfavored N‐terminal presequences and driven the evolution of internal targeting signals in MRPs.     

Molecular mechanism of co-translational protein targeting by the signal recognition particle

The signal recognition particle (SRP) is a key component of the cellular machinery that couples the ongoing synthesis of proteins to their proper localization, and has often served as a paradigm for understanding the molecular basis of protein localization within the cell. The SRP pathway exemplifies several key molecular events required for protein targeting to cellular membranes: the specific recognition of signal sequences on cargo proteins, the efficient delivery of cargo to the target membrane, the productive unloading of cargo to the translocation machinery and the precise spatial and temporal coordination of these molecular events. Here we highlight recent advances in our understanding of the molecular mechanisms underlying this pathway, and discuss new questions raised by these findings.     

A tale of two GTPases in cotranslational protein targeting

Guanosine triphosphatases (GTPases) comprise a superfamily of proteins that provide molecular switches to regulate numerous cellular processes. The "GTPase switch" paradigm, in which a GTPase acts as a bimodal switch that is turned "on" and "off" by external regulatory factors, has been used to interpret the regulatory mechanism of many GTPases. Recent work on a pair of GTPases in the signal recognition particle (SRP) pathway has revealed a distinct mode of GTPase regulation. Instead of the classical GTPase switch, the two GTPases in the SRP and SRP receptor undergo a series of conformational changes during their dimerization and reciprocal activation. Each conformational rearrangement provides a point at which these GTPases can communicate with and respond to their upstream and downstream biological cues, thus ensuring the spatial and temporal precision of all the molecular events in the SRP pathway. We suggest that the SRP and SRP receptor represent an emerging class of "multistate" regulatory GTPases uniquely suited to provide exquisite control over complex cellular pathways that require multiple molecular events to occur in a highly coordinated fashion.     

Heterologous signals allow efficient targeting of a nuclear-encoded fusion protein to plastids and endoplasmic reticulum in diverse plant species

Approximately 30% of plant nuclear genes appear to encode proteins targeted to the plastids or endoplasmic reticulum (ER). The signals that direct proteins into these compartments are diverse in sequence, but, on the basis of a limited number of tests in heterologous systems, they appear to be functionally conserved across species. To further test the generality of this conclusion, we tested the ability of two plastid transit peptides and an ER signal peptide to target green fluorescent protein (GFP) in 12 crops, including three monocots (barley, sugarcane, wheat) and nine dicots ( Arabidopsis , broccoli, cabbage, carrot, cauliflower, lettuce, radish, tobacco, turnip). In all species, transient assays following microprojectile bombardment or vacuum infiltration using Agrobacterium showed that the plastid transit peptides from tomato DCL (defective chloroplast and leaves) and tobacco RbcS [ribulose bisphosphate carboxylase (Rubisco) small subunit] genes were effective in targeting GFP to the leaf plastids. GFP engineered as a fusion to the N-terminal ER signal peptide from Arabidopsis basic chitinase and a C-terminal HDEL signal for protein retention in the ER was accumulated in the ER of all species. The results in tobacco were confirmed in stably transformed cells. These signal sequences should be useful to direct proteins to the plastid stroma or ER lumen in diverse plant species of biotechnological interest for the accumulation of particular recombinant proteins or for the modification of particular metabolic streams.     

Analysis of nuclear targeting activities of transport signals in the human immunodeficiency virus Rev protein

The human immunodeficiency Rev protein shuttles between the nucleus and cytoplasm, while accumulating to high levels in the nucleus. Rev has a nuclear localization signal (NLS; AA 35-50) with an arginine-rich motif (ARM) that interacts with importin beta and a leucine-rich nuclear export signal (NES; AA 75-84) recognized by CRM1/exportin 1. Here we explore nuclear targeting activities of the transport signals of Rev. GFP tagging and quantitative fluorescence microscopy were used to study the localization behavior of Rev NLS/ARM mutants under conditions inhibiting the export of Rev. Rev mutant M5 was actively transported to the nucleus, despite its known failure to bind importin beta. Microinjection of transport substrates with Rev-NES peptides revealed that the Rev-NES has both nuclear import and export activities. Replacement of amino acid residues "PLER" (77-80) of the NES with alanines abolished bidirectional transport activity of the Rev-NES. These results indicate that both transport signals of Rev have nuclear import capabilities and that the Rev NLS has more than one nuclear targeting activity. This suggests that Rev is able to use various routes for nuclear entry rather than depending on a single pathway.     

Functional implication of human serine/threonine kinase, hAIK, in cell cycle progression

Protein phosphorylation is involved in many biological activities and plays important roles in cell cycle progression. In the present study, we identified a serine/threonine kinase, hAIK, from human hepatic cells using degenerated polymerase chain reactions with a pair of primers derived from the highly conserved sequence in the catalytic domain of kinases. The full-length hAIK cDNA was then obtained, which contained 403 amino acids and was homologous to Drosophila Aurora2 and yeast Ipl1 proteins. Northern blotting analysis revealed that hAIK was highly expressed in the testis but not in other tissues. Expressions of hAIK drastically increased in cancer tissues/cell lines but not in fibroblasts or nontumorigenic cell lines. The recombinant hAIK protein phosphorylated itself and histone H1; this phosphorylation activity was totally abolished after a point mutation at the catalytic domain (hAIKm). During the interphase cell, hAIK was found mainly in the cytoplasm; during mitosis hAIK accumulated at the centrosomes. In addition, overexpression of hAIK in cancer cell lines (HEK293T and HeLa) appeared to inhibit cell cycle progression. None of these phenomena were observed in hAIKm whose kinase activity was rendered inactive. Our results suggest that hAIK protein/activity might modulate cell cycle progression by interacting with the centrosomes and/or proteins associated with these structures.     

Sorting pathway and molecular targeting signals for the Arabidopsis peroxin 3

Peroxin 3 (Pex3p) has been identified and characterized as a peroxisomal membrane protein in yeasts and mammals. We identified two putative homologs in Arabidopsis (AtPex3p, forms 1 and 2), both with an identical cluster of positively charged amino acid residues (RKHRRK) immediately preceding one of the two predicted transmembrane domains (TMD1). In transiently transformed Arabidopsis and tobacco BY-2 suspension-cultured cells, epitope-tagged AtPex3p (form 2) sorted post-translationally from the cytosol directly to peroxisomes, the first sorting pathway described for any peroxin in plants. TMD1 and RKHRRK were necessary for targeting form 2 to peroxisomes and sufficient for directing chloramphenicol acetyltransferase to peroxisomes in both cell types. The N and C termini of AtPex3p (form 2) extend into the peroxisomal matrix, different from mammal and yeast Pex3 proteins. Thus, two authentic peroxisomal membrane-bound Pex3p homologs possessing a membrane peroxisomal targeting signal, the first one defined for a plant peroxin and for any Pex3p homolog, exist in plant cells.     

信号识别颗粒(SRP)在膜蛋白定位中的作用

依赖信号识别颗粒(signal recognition particle,SRP)的共翻译转运是所有生命体中的一个保守途径,它将新生肽链的翻译与转运耦联在一起。超过30%的新合成的多肽链被SRP转运到正确位置。最近的研究表明,大肠杆菌中SRP抑制子可以规避SRP的需求。当SRP缺失时,翻译控制在介导膜蛋白定位方面起着关键作用。本综述总结了SRP底物如何在存在或缺失SRP的情况下转运到适当的位置以及翻译速率降低如何补偿SRP的缺失。我们还讨论了不同蛋白质对SRP的依赖程度。这一回顾将为进一步研究SRP功能及膜蛋白定位提供新思路。     

Identification of two distinct intracellular localization signals in STT3-B

The STT3 subunit of the oligosaccharyltransferase complex plays a critical role in the N-glycosylation process. From Arabidopsis thaliana to Homo sapiens, two functional STT3 isoforms have been identified, STT3-A and STT3-B. We report that the last transmembrane (TM) segment of STT3-B corresponds to a topogenic determinant that is sufficient for proper integration and orientation of STT3-B C-terminal domain. Notably, the last TM segment of STT3-A and -B isoforms present major differences in amino acid sequence and predicted 3D structure. We also identified a bipartite nuclear targeting sequence in the C-terminal tail of STT3-B that is absent in STT3-A. The latter sequence is sufficient to induce nucleolar localization of a reporter protein. Our results show that STT3-A and -B display two structural differences that may have a drastic influence on their function and might account for the remarkable evolutionary conservation of the two STT3 paralogs.     

Carboxyl-methylation of prenylated calmodulin CaM53 is required for efficient plasma membrane targeting of the protein

Prenylation is necessary for association of the petunia calmodulin CaM53 with the plasma membrane. To determine whether post-prenylation processing of the protein was also required for plasma membrane targeting, we studied the subcellular localization of a GFP-labelled CaM53 reporter in yeast and plant cells. Blocking of carboxyl-methylation of prenylated proteins either by a specific inhibitor or in mutant yeast cells resulted in localization of green fluorescence to what appears to be the endomembrane system, in contrast with the plasma membrane localization observed in control cells. We show that a prenyl-cysteine methyltransferase (PCM) activity that carboxyl-methylates prenylated CaM53 also exists in plant cells, and that it is required for efficient plasma membrane targeting. We also report an Arabidopsis gene with homology to PCM and demonstrate that it encodes a protein with PCM activity that localizes to the endomembrane system of plant cells, similar to prenylated but unmethylated CaM53. Together, our data suggest that, following prenylation, CaM53 is probably associated with the endomembrane system, where a PCM activity methylates the prenylated protein prior to targeting it to its final destination in the plasma membrane.     

Identification of a novel centrosomal protein CrpF46 involved in cell cycle progression and mitosis

A novel centrosome-related protein CrpF46 was detected using a serum F46 from a patient suffering from progressive systemic sclerosis. We identified the protein by immunoprecipitation and Western blotting followed by tandem mass spectrometry sequencing. The protein CrpF46 has an apparent molecular mass of ~60 kDa, is highly homologous to a 527 amino acid sequence of the C-terminal portion of the protein Golgin-245, and appears to be a splice variant of Golgin-245. Immunofluorescence microscopy of synchronized HeLa cells labeled with an anti-CrpF46 monoclonal antibody revealed that CrpF46 localized exclusively to the centrosome during interphase, although it dispersed throughout the cytoplasm at the onset of mitosis. Domain analysis using CrpF46 fragments in GFP-expression vectors transformed into HeLa cells revealed that centrosomal targeting is conferred by a C-terminal coiled-coil domain. Antisense CrpF46 knockdown inhibited cell growth and proliferation and the cell cycle typically stalled at S phase. The knockdown also resulted in the formation of poly-centrosomal and multinucleate cells, which finally became apoptotic. These results suggest that CrpF46 is a novel centrosome-related protein that associates with the centrosome in a cell cycle-dependent manner and is involved in the progression of the cell cycle and M phase mechanism.     

R3F, a novel membrane-associated glycogen targeting subunit of protein phosphatase 1 regulates glycogen synthase in astrocytoma cells in response to glucose and extracellular signals

Abnormal regulation of brain glycogen metabolism is believed to underlie insulin-induced hypoglycaemia, which may be serious or fatal in diabetic patients on insulin therapy. A key regulator of glycogen levels is glycogen targeted protein phosphatase 1 (PP1), which dephosphorylates and activates glycogen synthase (GS) leading to an increase in glycogen synthesis. In this study, we show that the gene PPP1R3F expresses a glycogen-binding protein (R3F) of 82.8 kDa, present at the high levels in rodent brain. R3F binds to PP1 through a classical 'RVxF' binding motif and substitution of Phe39 for Ala in this motif abrogates PP1 binding. A hydrophobic domain at the carboxy-terminus of R3F has similarities to the putative membrane binding domain near the carboxy-terminus of striated muscle glycogen targeting subunit G(M)/R(GL), and R3F is shown to bind not only to glycogen but also to membranes. GS interacts with PP1-R3F and is hyperphosphorylated at glycogen synthase kinase-3 sites (Ser640 and Ser644) when bound to R3F(Phe39Ala). Deprivation of glucose or stimulation with adenosine or noradrenaline leads to an increased phosphorylation of PP1-R3F bound GS at Ser640 and Ser644 curtailing glycogen synthesis and facilitating glycogen degradation to provide glucose in astrocytoma cells. Adenosine stimulation also modulates phosphorylation of R3F at Ser14/Ser18.     

Identification and characterization of two morphologically distinct stem cell subpopulations from human urine samples

Urine-derived stem cells(USCs) have shown potentials for the treatment of skeletal and urological disorders. Based on published literature and our own data, USCs consist of heterogeneous populations of cells. In this paper, we identify and characterize two morphologically distinct subpopulations of USCs from human urine samples, named as spindle-shaped USCs(SS-USCs) and rice-shaped USCs(RS-USCs) respectively. The two subpopulations showed similar clone-forming efficiency, while SS-USCs featured faster proliferation, higher motility, and greater potential for osteogenic and adipogenic differentiation, RS-USCs showed greater potential for chondrogenic differentiation. POU5F1 was strongly expressed in both subpopulations, but MYC was weakly expressed. Both subpopulations showed similar patterns of CD24, CD29, CD34, CD44, CD73, CD90 and CD105 expression, while a higher percentage of RS-USCs were positive for CD133. SS-USCs were positive for VIM, weakly positive for SLC12A1 and UMOD, and negative for KRT18, NPHS1, AQP1 and AQP2, indicating a renal mesenchyme origin; while RSUSCs are positive for VIM, partially positive for KRT18, NPHS1, AQP1, SLC12A1 and UMOD, and negative for AQP2,indicating a nephron tubule origin. The above results can facilitate understanding of the biological characteristics of subpopulations of USCs, and provide a basis for further research and applications of such cells.     

Isolation and characterization of the two distinct genes for human glutamate dehydrogenase

Two genomic clones containing a part of the glutamate dehydrogenase gene were isolated from a human genomic library. The restriction map of both clones were distinctly different from one another, although the nucleotide sequences of the three exons that they contained were virtually the same in each clone. Southern blotting analysis of the genomic DNAs from several unrelated human individuals revealed that in every case the probe hybridized with at least two DNA fragments of different sizes, each characteristic to one of the two clones. These results strongly suggest that the two clones presently obtained do not result from polymorphism but are generated from two different gene loci for glutamate dehydrogenase on the human chromosome.     

Regulation of cell contraction and membrane ruffling by distinct signals in migratory cells.

Cell migration and wound contraction requires assembly of actin into a functional myosin motor unit capable of generating force. However, cell migration also involves formation of actin-containing membrane ruffles. Evidence is provided that actin-myosin assembly and membrane ruffling are regulated by distinct signaling pathways in the migratory cell. Interaction of cells with extracellular matrix proteins or cytokines promote cell migration through activation of the MAP kinases ERK1 and ERK2 as well as the molecular coupling of the adaptor proteins p130CAS and c-CrkII. ERK signaling is independent of CAS/Crk coupling and regulates myosin light chain phosphorylation leading to actin-myosin assembly during cell migration and cell-mediated contraction of a collagen matrix. In contrast, membrane ruffling, but not cell contraction, requires Rac GTPase activity and the formation of a CAS/Crk complex that functions in the context of the Rac activating protein DOCK180. Thus, during cell migration ERK and CAS/Crk coupling operate as components of distinct signaling pathways that control actin assembly into myosin motors and membrane ruffles, respectively.     

Only one of the two signals required for initiation of the cell cycle is associated with cellular accumulation of ribosomal RNA

Two intracellular functions are elicited by the serum component in culture media in order to initiate the cell cycle: "competence" and "progression". Although both functions have to be present simultaneously for start of cell division, it is shown here for Swiss 3T3 cells that only one of them, the progression signal, is associated with reaccumulation of ribosomal RNA. This result points to the cellular complement of ribosomes as one of the limiting parameters for cell division.     

Molecular characterization of caveolin association with the Golgi complex: identification of a cis-Golgi targeting domain in the caveolin molecule.

Caveolins are integral membrane proteins which are a major component of caveolae. In addition, caveolins have been proposed to cycle between intracellular compartments and the cell surface but the exact trafficking route and targeting information in the caveolin molecule have not been defined. We show that antibodies against the caveolin scaffolding domain or against the COOH terminus of caveolin-1 show a striking specificity for the Golgi pool of caveolin and do not recognize surface caveolin by immunofluorescence. To analyze the Golgi targeting of caveolin in more detail, caveolin mutants were expressed in fibroblasts. Specific mutants lacking the NH2 terminus were targeted to the cis Golgi but were not detectable in surface caveolae. Moreover, a 32-amino acid segment of the putative COOH-terminal cytoplasmic domain of caveolin-3 was targeted specifically and exclusively to the Golgi complex and could target a soluble heterologous protein, green fluorescent protein, to this compartment. Palmitoylation-deficient COOH-terminal mutants showed negligible association with the Golgi complex. This study defines unique Golgi targeting information in the caveolin molecule and identifies the cis Golgi complex as an intermediate compartment on the caveolin cycling pathway.     

A centrosomal function for the human Nek2 protein kinase, a member of the NIMA family of cell cycle regulators.

Nek2, a mammalian protein kinase of unknown function, is closely related to the mitotic regulator NIMA of Aspergillus nidulans. Here we show by both immunofluorescence microscopy and biochemical fractionation that human Nek2 localizes to the centrosome. Centrosome association occurs throughout the cell cycle, including all stages of mitosis, and is independent of microtubules. Overexpression of active Nek2 induces a striking splitting of centrosomes, whereas prolonged expression of either active or inactive Nek2 leads to dispersal of centrosomal material and loss of a focused microtubule-nucleating activity. Surprisingly, this does not prevent entry into mitosis, as judged by the accumulation of mitotically arrested cells induced by co-expression of a non-destructible B-type cyclin. These results bear on the dynamic function of centrosomes at the onset of mitosis. Moreover, they indicate that one function of mammalian Nek2 relates to the centrosome cycle and thus provide a new perspective on the role of NIMA-related kinases.     

Molecular and cellular characterization of the Down syndrome critical region protein 2

Down syndrome (DS) is caused by trisomy for human chromosome 21 and is the most common genetic cause of mental retardation. The distal 10 Mb region of the long arm of the chromosome has been proposed to be associated with many of the abnormalities seen in DS. This region is often referred to as the Down syndrome critical region (DSCR). We report here the results of our analyses of the DSCR protein 2 (DSCR2). Results from transiently transfected COS-1 and HEK293 cells suggest that DSCR2 is synthesized as a 43 kDa precursor protein, from which the N-terminus is cleaved resulting in a polypeptide of 41 kDa. The polypeptide is modified by still uncharacterized co- or post-translational modifications increasing the predicted molecular weight of 32.8 kDa by about 10 kDa. Analyses of the only putative N-glycosylation site by in vitro mutagenesis excluded the possibility of the contribution of N-glycosylation to this increase in molecular weight. Further, the results of intracellular localization studies and membrane fractionation assays indicate that DSCR2 is targeted to a cytoplasmic compartment as a soluble form.