Rtt107 BRCT domains act as a targeting module in the DNA damage response

Cells are constantly exposed to assaults that cause DNA damage, which must be detected and repaired to prevent genome instability. The DNA damage response is mediated by key kinases that activate various signaling pathways. In Saccharomyces cerevisiae, one of these kinases is Mec1, which phosphorylates numerous targets, including H2A and the DNA damage protein Rtt107. In addition to being phosphorylated, Rtt107 contains six BRCA1 C-terminal (BRCT) domains, which typically recognize phospho-peptides. Thus Rtt107 represented an opportunity to study complementary aspects of the phosphorylation cascades within one protein. Here we sought to describe the functional roles of the multiple BRCT domains in Rtt107. Rtt107 BRCT5/6 facilitated recruitment to sites of DNA lesions via its interaction with phosphorylated H2A. Rtt107 BRCT3/4 also contributed to Rtt107 recruitment, but BRCT3/4 was not sufficient for recruitment when BRCT5/6 was absent. Intriguingly, both mutations that affected Rtt107 recruitment also abrogated its phosphorylation. Pointing to its modular nature, replacing Rtt107 BRCT5/6 with the BRCT domains from the checkpoint protein Rad9 was able to sustain Rtt107 function. Although Rtt107 physically interacts with both the endonuclease Slx4 and the DNA replication and repair protein Dpb11, only Slx4 was dependent on Rtt107 for its recruitment to DNA lesions. Fusing Rtt107 BRCT5/6 to Slx4, which presumably allows artificial recruitment of Slx4 to DNA lesions, alleviated some phenotypes of rtt107Δ mutants, indicating the functional importance of Slx4 recruitment. Together this data revealed a key function of the Rtt107 BRCT domains for targeting of both itself and its interaction partners to DNA lesions.     

Vacuolar two-pore K+ channels act as vacuolar osmosensors

? Plant two-pore K(+) channels (TPKs) have been shown previously to play a role in vacuolar K(+) homeostasis. TPK activity is insensitive to membrane voltage, but regulated by cytoplasmic calcium and 14-3-3 proteins. This study reports that membrane stretch and osmotic gradients also alter the activity of TPKs from Arabidopsis, rice and barley, and that this may have a physiological relevance for osmotic homeostasis. ? Mechanosensitivity was studied using patch clamp experiments and TPKs from Arabidopsis, rice and barley. In addition, the capability of TPKs to act as osmosensors was determined. By using protoplast disruption assays and intact plant survival assays, in genotypes that differed in TPK expression, the physiological relevance of TPK-based osmosensing was tested. ? TPKs from all three species showed varying degrees of mechanosensitivity. TPK activity in channels from all three species was sensitive to trans-tonoplast osmotic gradients. TPK osmosensing is likely to proceed via the detection of small perturbations in membrane tension. Intact plant and protoplast assays showed that TPK-based osmosensing is important during exposure to rapid changes in external osmolarity. ? Vacuolar TPK channels can act as intracellular osmosensors and rapidly increase channel activity during hypo-osmotic shock to release vacuolar K(+) .     

Independent mutations at the amino terminus of a protein act as surrogate signals for mitochondrial import.

Intracellular delivery of the mitochondrial F1-ATPase beta-subunit precursor from the cytoplasm into the matrix of mitochondria is prevented by deletion of its mitochondrial import signal, a basic amphipathic alpha-helix at its amino terminus. Using a complementation assay, we have selected spontaneous mutations which restore the correct in vivo localization of the protein containing the import signal deletion. Analysis of these mutations revealed that different functional surrogate mitochondrial targeting signals formed within a narrow region of the extreme amino terminus of the import signal deleted beta-subunit. These modifications specifically replace different acidic residues with neutral or basic residues to generate a less acidic amphipathic helix within a region of the protein which is accessible for interaction with the membrane surface. The observations of this study confirm the requirement for amphipathicity as part of the mitochondrial import signal and suggest how mitochondrial targeting signals may have evolved within the extreme amino terminus of mitochondrial proteins.     

A new class of lysosomal/vacuolar protein sorting signals

A number of inherited lysosomal diseases are known to result from missorting of lysosomal proteins. Considerable attention has been directed toward an understanding of this sorting pathway, and it has become apparent that different mechanisms are used for the sorting of lysosomal membrane and soluble proteins. Protein sorting to the yeast vacuole/lysosome provides a simple model system to study this process. We have mapped the first sorting signal in a vacuolar membrane protein, repressible alkaline phosphatase, and have shown it to be both necessary and sufficient for vacuolar delivery of this enzyme. The sorting information is confined to the transmembrane and cytoplasmic tail region of this type II integral membrane protein. The location of this sorting signal provides an explanation for some of the differences observed between membrane and soluble vacuolar protein sorting.     

Different domains of the M-band protein myomesin are involved in myosin binding and M-band targeting

Myomesin is a 185-kDa protein located in the M-band of striated muscle where it interacts with myosin and titin, possibly connecting thick filaments with the third filament system. By using expression of epitope-tagged myomesin fragments in cultured cardiomyocytes and biochemical binding assays, we could demonstrate that the M-band targeting activity and the myosin-binding site are located in different domains of the molecule. An N-terminal immunoglobulin-like domain is sufficient for targeting to the M-band, but solid-phase overlay assays between individual N-terminal domains and the thick filament protein myosin revealed that the unique head domain contains the myosin-binding site. When expressed in cardiomyocytes, the head domains of rat and chicken myomesin showed species-specific differences in their incorporation pattern. The head domain of rat myomesin localized to a central area within the A-band, whereas the head domain of chicken myomesin was diffusely distributed in the cytoplasm. We therefore conclude that the head domain of myomesin binds to myosin but that this affinity is not sufficient for the restriction of the domain to the M-band in vivo. Instead, the neighboring immunoglobulin-like domain is essential for the precise incorporation of myomesin into the M-band, possibly because of interaction with a yet unknown protein of the sarcomere.     

Advances in the prediction of protein targeting signals

Enlarged sets of reference data and special machine learning approaches have improved the accuracy of the prediction of protein subcellular localization. Recent approaches report over 95% correct predictions with low fractions of false-positives for secretory proteins. A clear trend is to develop specifically tailored organism- and organelle-specific prediction tools rather than using one general method. Focus of the review is on machine learning systems, highlighting four concepts: the artificial neural feed-forward network, the self-organizing map (SOM), the Hidden-Markov-Model (HMM), and the support vector machine (SVM).     

Interaction of mitochondrial targeting signals with acidic receptor domains along the protein import pathway: evidence for the ''acid chain'' hypothesis.

Mitochondrial precursor proteins with basic targeting signals may be transported across the outer membrane by sequential binding to acidic receptor sites of increasing affinity. To test this 'acid chain' hypothesis, we assayed the interaction of mitochondrial precursors with three acidic receptor domains: the cytosolic domain of Tom20 and the intermembrane space domain of Tom22 and Tim23. The apparent affinity and salt resistance of precursor binding increased in the order Tom20<Tom22 (internal)<Tim23. Precursor binding to the three acidic receptor domains and to the pure cytosolic domain of Tom70 was inhibited by excess targeting peptide, but not by an equally basic control peptide. In this membrane-free and defined system, a precursor pre-bound to the Tom70 or Tom20 domain was transferred efficiently to the Tim23 domain. Transfer was stimulated by the internal Tom22 domain and was much less efficient in the reverse direction. Precursors destined for the outer membrane bound only to Tom20, but not to the internal Tom22 or the Tim23 domain, and a precursor destined for the inner membrane bound only to the Tom20 and the internal Tom22 domain, but not to the Tim23 domain. These results suggest that specific and sequential binding of a targeting signal to strategically situated acidic receptors delivers a precursor across the outer membrane and contributes to intramitochondrial sorting of imported proteins.     

Prediction of organellar targeting signals.

The subcellular location of a protein is an important characteristic with functional implications, and hence the problem of predicting subcellular localization from the amino acid sequence has received a fair amount of attention from the bioinformatics community. This review attempts to summarize the present state of the art in the field.     

Membrane protein spanning segments as export signals.

Escherichia coli lac permease is a polytopic integral membrane protein with six translocated (periplasmic) domains. Individual N-terminal cytoplasmic regions and membrane-spanning segments adjacent to each of the periplasmic domains acted as export signals for an attached sensor protein (alkaline phosphatase). However, the export activity of one of the spanning segments was considerably lower than that of the others, and was limited by the presence of a positively charged residue (Arg302). These observations are compatible with models of membrane protein insertion in which hydrophilic domains are translocated independently. However, the results suggest that efficient translocation may sometimes require interaction between individual spanning segments.     

Peroxisomal protein targeting and identification of peroxisomal targeting signals in cholesterol biosynthetic enzymes

At least three different subcellular compartments, including peroxisomes, are involved in cholesterol synthesis. Recently, it has been demonstrated that peroxisomes contain a number of enzymes involved in cholesterol biogenesis that previously were considered to be cytosolic or located in the endoplasmic reticulum. Peroxisomes have been shown to contain acetoacetyl-CoA thiolase, HMG-CoA synthase, HMG-CoA reductase, mevalonate kinase, phosphomevalonate kinase, phosphomevalonate decarboxylase, isopentenyl diphosphate isomerase and FPP synthase. Moreover, the activities of these enzymes are also significantly decreased in liver tissue and fibroblast cells obtained from patients with peroxisomal deficiency diseases. In addition, the cholesterol biosynthetic capacity is severely impaired in cultured skin fibroblasts obtained from patients with peroxisomal deficiency diseases. These findings support the proposal that peroxisomes play an essential role in isoprenoid biosynthesis. This paper presents a review of peroxisomal protein targeting and of recent studies demonstrating the localization of cholesterol biosynthetic enzymes in peroxisomes and the identification of peroxisomal targeting signals in these proteins.     

Interaction of a potential vacuolar targeting receptor with amino- and carboxyl-terminal targeting determinants.

A protein of 80 kD from developing pea (Pisum sativum) cotyledons has previously been shown to exhibit characteristics of a vacuolar targeting receptor by means of its affinity for the amino-terminal vacuolar targeting sequence of proaleurain from barley (Hordeum vulgare). In this report we show that the same protein also binds to the amino-terminal targeting peptide of prosporamin from sweet potato (Ipomoea batatas) and to the carboxyl-terminal targeting determinant of pro-2S albumin from Brazil nut (Bertholletia excelsa). The receptor protein does not bind to the carboxyl-terminal propeptide (representing the targeting sequence) of barley lectin. The binding of the 80-kD protein to the sporamin determinant involves a motif (NPIR) that has been shown to be crucial for vacuolar targeting in vivo. The binding to the carboxyl-terminal targeting determinant of pro-2S albumin appears to involve the carboxyl-terminal propeptide and the adjacent five amino acids of the mature protein. The 80-kD protein does not bind to peptide sequences that have been shown to be incompetent in directing vacuolar targeting.     

Distinct targeting and fusion functions of the PX and SNARE domains of yeast vacuolar Vam7p

Regulated membrane fusion requires organelle tethering, enrichment of selected proteins and lipids at the fusion site, bilayer distortion, and lipid rearrangement. Yeast vacuole homotypic fusion requires regulatory lipids (ergosterol, diacylglycerol, and phosphoinositides), the Rab family GTPase Ypt7p, the multisubunit Ypt7p-effector complex HOPS (homotypic fusion and vacuole protein sorting), and four SNAREs. One SNARE, Vam7p, has an N-terminal PX domain which binds to phosphatidylinositol 3-phosphate (PI(3)P) and to HOPS and a C-terminal SNARE domain but no apolar membrane anchor. We have exploited an in vitro reaction of vacuole fusion to analyze the functions of each domain, removing the PX domain or mutating it to abolish its PI(3)P affinity. Lowering the PI(3)P affinity of the PX domain, or even deleting the PX domain, affects the fusion K(m) for Vam7p but not the maximal fusion rate. Fusion driven by the SNARE domain alone is strikingly enhanced by the PLC inhibitor U73122 through enhanced binding of Vam7p SNARE domain to vacuoles, and the further addition of Plc1p blocks this U73122 effect. The PX domain, through its affinities for phosphoinositides and HOPS, is thus exclusively required for enhancing the targeting of Vam7p rather than for execution of the Vam7p functions in HOPS.SNARE complex assembly and fusion.     

Receptor salvage from the prevacuolar compartment is essential for efficient vacuolar protein targeting

We have characterized the requirements to inhibit the function of the plant vacuolar sorting receptor BP80 in vivo and gained insight into the crucial role of receptor recycling between the prevacuolar compartment and the Golgi apparatus. The drug wortmannin interferes with the BP80-mediated route to the vacuole and induces hypersecretion of a soluble BP80-ligand. Wortmannin does not prevent receptor-ligand binding itself but causes BP80 levels to be limiting. Consequently, overexpression of BP80 partially restores vacuolar cargo transport. To simulate receptor traffic, we tested a truncated BP80 derivative in which the entire lumenal domain of BP80 has been replaced by the green fluorescent protein (GFP). The resulting chimeric protein (GFP-BP80) accumulates in the prevacuolar compartment as expected, but a soluble GFP fragment can also be detected in purified vacuoles. Interestingly, GFP-BP80 coexpression interferes with the correct sorting of a BP80-ligand and causes hypersecretion that is reversible by expressing a 10-fold excess of full-length BP80. This suggests that GFP-BP80 competes with endogenous BP80 mainly at the retrograde transport route that rescues receptors from the prevacuolar compartment. Treatment with wortmannin causes further leakage of GFP-BP80 from the prevacuolar compartment to the vacuoles, whereas BP80-ligands are secreted. We propose that recycling of the vacuolar sorting receptor from the prevacuolar compartment to the Golgi apparatus is an essential process that is saturable and wortmannin sensitive.     

Distinct protein domains regulate ciliary targeting and function of C. elegans PKD-2

TRPP2 (transient receptor potential polycystin-2) channels function in a range of cells where they are localized to specific subcellular regions including the endoplasmic reticulum (ER) and primary cilium. In humans, TRPP2/PC-2 mutations severely compromise kidney function and cause autosomal dominant polycystic kidney disease (ADPKD). The Caenorhabditis elegans TRPP2 homolog, PKD-2, is restricted to the somatodendritic (cell body and dendrite) and ciliary compartments of male specific sensory neurons. Within these neurons PKD-2 function is required for sensation. To understand the mechanisms regulating TRPP2 subcellular distribution and activity, we performed in vivo structure-function-localization studies using C. elegans as a model system. Our data demonstrate that somatodendritic and ciliary targeting requires the transmembrane (TM) region of PKD-2 and that the PKD-2 cytosolic termini regulate subcellular distribution and function. Within neuronal cell bodies, PKD-2 colocalizes with the OSM-9 TRP vanilloid (TRPV) channel, suggesting that these TRPP and TRPV channels may function in a common process. When human TRPP2/PC-2 is heterologously expressed in transgenic C. elegans animals, PC-2 does not visibly localize to cilia but does partially rescue pkd-2 null mutant defects, suggesting that human PC-2 and PKD-2 are functional homologs.     

Tonoplast intrinsic protein isoforms as markers for vacuolar functions

Plant cell vacuoles may have storage or lytic functions, but biochemical markers specific for the tonoplasts of functionally distinct vacuoles are poorly defined. Here, we use antipeptide antibodies specific for the tonoplast intrinsic proteins alpha-TIP, gamma-TIP, and delta-TIP in confocal immunofluorescence experiments to test the hypothesis that different TIP isoforms may define different vacuole functions. Organelles labeled with these antibodies were also labeled with antipyrophosphatase antibodies, demonstrating that regardless of their size, they had the expected characteristics of vacuoles. Our results demonstrate that the storage vacuole tonoplast contains delta-TIP, protein storage vacuoles containing seed-type storage proteins are marked by alpha- and delta- or alpha- and delta- plus gamma-TIP, whereas vacuoles storing vegetative storage proteins and pigments are marked by delta-TIP alone or delta- plus gamma-TIP. In contrast, those marked by gamma-TIP alone have characteristics of lytic vacuoles, and results from other researchers indicate that alpha-TIP alone is a marker for autophagic vacuoles. In root tips, relatively undifferentiated cells that contain vacuoles labeled separately for each of the three TIPs have been identified. These results argue that plant cells have the ability to generate and maintain three separate vacuole organelles, with each being marked by a different TIP, and that the functional diversity of the vacuolar system may be generated from different combinations of the three basic types.     

N-glycans as apical targeting signals in polarized epithelial cells

MDCK (Madin-Darby canine kidney) cells represent a good model of polarized epithelium to investigate the signals involved in the apical targeting of proteins. As reported previously, GPI (glycosylphosphatidylinositol) anchors mediate the apical sorting of proteins in polarized epithelial cells through their interaction with lipid rafts. However, using a naturally N-glycosylated and GPI-anchored protein, we found that the GPI anchor does not influence the targeting of the protein. It is, in fact, the N-glycans that signal the protein to the apical surface. In the present review, the role of N-glycans and GPI anchors as apical signals is discussed along with the putative mechanisms involved.     

Cytoplasmic tail of coronavirus spike protein has intracellular targeting signals

Intracellular trafficking and localization studies of spike protein from SARS and OC43 showed that SARS spike protein is localized in the ER or ERGIC compartment and OC43 spike protein is predominantly localized in the lysosome. Differential localization can be explained by signal sequence. The sequence alignment using Clustal W shows that the signal sequence present at the cytoplasmic tail plays an important role in spike protein localization. A unique GYQEL motif is identified at the cytoplasmic terminal of OC43 spike protein which helps in localization in the lysosome, and a novel KLHYT motif is identified in the cytoplasmic tail of SARS spike protein which helps in ER or ERGIC localization. This study sheds some light on the role of cytoplasmic tail of spike protein in cell-to-cell fusion, coronavirus host cell fusion and subsequent pathogenicity.