386 research outputs found
Intrinsic tethering activity of endosomal Rab proteins.
Rab small G proteins control membrane trafficking events required for many processes including secretion, lipid metabolism, antigen presentation and growth factor signaling. Rabs recruit effectors that mediate diverse functions including vesicle tethering and fusion. However, many mechanistic questions about Rab-regulated vesicle tethering are unresolved. Using chemically defined reaction systems, we discovered that Vps21, a Saccharomyces cerevisiae ortholog of mammalian endosomal Rab5, functions in trans with itself and with at least two other endosomal Rabs to directly mediate GTP-dependent tethering. Vps21-mediated tethering was stringently and reversibly regulated by an upstream activator, Vps9, and an inhibitor, Gyp1, which were sufficient to drive dynamic cycles of tethering and detethering. These experiments reveal a previously undescribed mode of tethering by endocytic Rabs. In our working model, the intrinsic tethering capacity Vps21 operates in concert with conventional effectors and SNAREs to drive efficient docking and fusion
Sumoylation of The Budding Yeast Kinetochore Protein Ndc10 is Required for Ndc10 Spindle Localization and Regulation of Anaphase Spindle Elongation
Posttranslational modification by the ubiquitin-like protein SUMO (small ubiquitin-like modifier) is emerging as an important regulator in many cellular processes, including genome integrity. In this study, we show that the kinetochore proteins Ndc10, Bir1, Ndc80, and Cep3, which mediate the attachment of chromosomes to spindle microtubules, are sumoylated substrates in budding yeast. Furthermore, we show that Ndc10, Bir1, and Cep3 but not Ndc80 are desumoylated upon exposure to nocodazole, highlighting the possibility of distinct roles for sumoylation in modulating kinetochore protein function and of a potential link between the sumoylation of kinetochore proteins and mitotic checkpoint function. We find that lysine to arginine mutations that eliminate the sumoylation of Ndc10 cause chromosome instability, mislocalization of Ndc10 from the mitotic spindle, abnormal anaphase spindles, and a loss of Bir1 sumoylation. These data suggest that sumoylation of Ndc10 and other kinetochore proteins play a critical role during the mitotic process
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Comparative analysis of Saccharomyces cerevisiae WW domains and their interacting proteins
BACKGROUND: The WW domain is found in a large number of eukaryotic proteins implicated in a variety of cellular processes. WW domains bind proline-rich protein and peptide ligands, but the protein interaction partners of many WW domain-containing proteins in Saccharomyces cerevisiae are largely unknown. RESULTS: We used protein microarray technology to generate a protein interaction map for 12 of the 13 WW domains present in proteins of the yeast S. cerevisiae. We observed 587 interactions between these 12 domains and 207 proteins, most of which have not previously been described. We analyzed the representation of functional annotations within the network, identifying enrichments for proteins with peroxisomal localization, as well as for proteins involved in protein turnover and cofactor biosynthesis. We compared orthologs of the interacting proteins to identify conserved motifs known to mediate WW domain interactions, and found substantial evidence for the structural conservation of such binding motifs throughout the yeast lineages. The comparative approach also revealed that several of the WW domain-containing proteins themselves have evolutionarily conserved WW domain binding sites, suggesting a functional role for inter- or intramolecular association between proteins that harbor WW domains. On the basis of these results, we propose a model for the tuning of interactions between WW domains and their protein interaction partners. CONCLUSION: Protein microarrays provide an appealing alternative to existing techniques for the construction of protein interaction networks. Here we built a network composed of WW domain-protein interactions that illuminates novel features of WW domain-containing proteins and their protein interaction partners
Interaction of an atypical Plasmodium falciparum ETRAMP with human apolipoproteins
Background: In order to establish a successful infection in the human host, the malaria parasite Plasmodium falciparum must establish interactions with a variety of human proteins on the surface of different cell types, as well as with proteins inside the host cells. To better understand this aspect of malaria pathogenesis, a study was conducted with the goal of identifying interactions between proteins of the parasite and those of its human host.
Methods: A modified yeast two-hybrid methodology that preferentially selects protein fragments that can be expressed in yeast was used to conduct high-throughput screens with P. falciparum
protein fragments against human liver and cerebellum libraries. The resulting dataset was analyzed to exclude interactions that are not likely to occur in the human host during infection.
Results: An initial set of 2,200 interactions was curated to remove proteins that are unlikely to play a role in pathogenesis based on their annotation or localization, and proteins that behave promiscuously in the two-hybrid assay, resulting in a final dataset of 456 interactions. A cluster that
implicates binding between P. falciparum PFE1590w/ETRAMP5, a putative parasitophorous vacuole membrane protein, and human apolipoproteins ApoA, ApoB and ApoE was selected for further analysis. Different isoforms of ApoE, which are associated with different outcomes of malaria infection, were shown to display differential interactions with PFE1590w.
Conclusion: A dataset of interactions between proteins of P. falciparum and those of its human host was generated. The preferential interaction of the P. falciparum PFE1590w protein with the
human ApoE e3 and ApoE e4 isoforms, but not the ApoE e2 isoform, supports the hypothesis that ApoE genotype affects risk of malaria infection. The dataset contains other interactions of potential
relevance to disease that may identify possible vaccine candidates and drug targets.This work was supported in part by grant P50 GM64655 from the NIH
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