Degradation of membrane proteins in the vacuole/lysosome is dependent on their prior sorting into the multivesicular body (MVB) pathway. This sorting process involves incorporation of proteins into vesicles that are formed by budding of the limiting membrane of the endosome into the lumen of the organelle. The MVB sorting process on the whole is highly conserved from yeast to human, and depends on the Vps27/Hrs, ESCRT-I, -II, and -III protein complexes functioning sequentially on the endosomal membrane, as well as on additional factors, such as the ubiquitinating enzyme Rsp5/Nedd4. It has now been established that ubiquitin serves as a sorting signal for many cargoes into the MVB pathway. <p>In this thesis work, we provide evidence that Bro1 is not required for protein ubiquitination or early steps of endocytosis, but functions at the late endosome level as an integral component of the MVB pathway. Similarly to its human homologue Alix, Bro1 interacts with components of the ESCRT-I and ESCRT-III complexes. The putative role of Bro1/Alix in bridging an interaction between ESCRT-I and –III might be important to strengthen an association of these protein complexes to allow efficient sorting of cargo proteins. Deficiency in Bro1 results in recycling of the endocytosed Gap1 permease back to the plasma membrane, a process coupled to deubiquitination of the permease. This recycling is a non-classical phenotype for cells impaired in MVB pathway thus suggesting Bro1 to have a particular role in this sorting process. Furthermore, the conserved C-terminal proline-rich domain (PRD) of Bro1 is specifically important for MVB sorting of cargo proteins that are subject to ubiquitination. We show Bro1 (via its PRD) to play a highly important role in recruitment of the deubiquitinating enzyme Doa4 to the endosome. Consistent with this, Bro1 is required for deubiquitination of cargo proteins, a step occurring just before cargo incorporation into the endosomal vesicles, and similarly to Doa4, for ubiquitin recycling. In contrast to previous interpretations, we show that Doa4 has a direct role in sorting of ubiquitinated cargo proteins into the MVB pathway. We propose that Doa4 – via its association to Bro1 - achieves this role by catalyzing deubiquitination of cargo proteins and/or some components of the MVB sorting machinery. We further show Bro1 to interact with the ubiquitin ligase Rsp5, which, in addition to being required for cargo protein ubiquitination at the plasma membrane, apparently contributes to multiple steps of endocytosis and MVB sorting. Also the Bro1-Rsp5 interaction is dependent on the C-terminal PRD region of Bro1. We propose that this interaction is conserved. <p>A role for ubiquitin in regulation of the MVB sorting machinery is emerging: the function of factors recognizing and sorting ubiquitinated cargo proteins in the MVB pathway is suggested to be coupled to their cycling between ubiquitinated and deubiquitinated stages. A growing body of evidence indicates that ubiquitin ligases of the Rsp5/Nedd4 family play a central role in this regulation. We speculate the Bro1/Alix protein, through its ability to simultaneously interact with factors of the MVB sorting machinery and with ubiquitinating and deubiquitinating enzymes to play a central role in the successive rounds of ubiquitination and deubiquitination of specific factors along the MVB pathway. <p> / Doctorat en sciences, Spécialisation biologie moléculaire / info:eu-repo/semantics/nonPublished

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Stomata are small pores that are located on the surface of epidermal leaves, and they can regulate the uptake of CO2 and prevent water lose by opening and closing the pores. Stomata of plants can be regulated by external condition such as CO2, biotic and abiotic stresses and internal factors. CAM (crassulacean acid metabolism) plants adapt to hot and dry environments by closing stomata during the day and opening stomata during the cool night. However, it is still unclear how CAM plants open their stomata during the night and close them during the day. In this study, a number of factors were evaluated for their potential roles in promoting stomatal opening in the model CAM plant Kalanchoe laxiflora. Citrate is an important organic acid and it accumulates during the night in CAM plants. It is shown in this study that citrate promoted stomatal opening in detached leaf epidermis of Kalanchoe laxiflora. Further, the cytokinin zeatin is also shown to stimulate stomatal opening in detached leave of Kalanchoe laxiflora. Melatonin is an important regulator of circadian rhythms in mammals and has been implicated in regulation of plant abiotic stress responses. Melatonin was detected in the leaves of Kalanchoe laxiflora. It promoted stomatal opening in detached epidermis of Kalanchoe laxiflora. Together, these results suggest that stomata of Kalanchoe laxiflora respond to citrate and malate which are the main organic acids accumulate during nighttime and also to some signaling molecules (zeatin, melatonin, and serotonin) by opening stomata during dark period.

Indiana University-Purdue University Indianapolis (IUPUI) / Patients with the genetic disorder tuberous sclerosis complex (TSC) suffer from neoplastic growths in multiple organ systems. These growths are the result of inactivating mutations in either the TSC1 or TSC2 tumor suppressor genes, which negatively regulate the activity of mammalian target of rapamycin complex 1(mTORC1). There is currently no cure for this disease; however, my research has found that cells harboring TSC2-inactivating mutations derived from a rat model of TSC are sensitive to apoptosis induced by the clinically approved proteasome inhibitor, bortezomib, in a manner dependent on their high levels of mTORC1 activation. We see that bortezomib induces the unfolded protein response (UPR) in our cell model of TSC, resulting in cell death via apoptosis. The UPR is induced by accumulation of unfolded protein in the endoplasmic reticulum (ER) which activates the three branches of this pathway: Activating transcription factor 6 (ATF6) cleavage, phosphorylation of eukaryotic initiation factor 2α (eIF2α), and the splicing of X-box binding protein1 (XBP1) mRNA. Phosphorylation of eIF2α leads to global inhibition of protein synthesis, preventing more unfolded protein from accumulating in the ER. This phosphorylation also induces the transcription and translation of ATF4 and CCAAT-enhancer binding protein homologous protein (CHOP). Blocking mTORC1 activity in these cells using the mTORC1 inhibitor, rapamycin, prevented the expression of ATF4 and CHOP at both the mRNA and protein level during bortezomib treatment. Rapamycin treatment also reduced apoptosis induced by bortezomib; however, it did not affect bortezomib-induced eIF2α phosphorylation or ATF6 cleavage. These data indicate that rapamycin can repress the induction of UPR-dependent apoptosis by suppressing the transcription of ATF4 and CHOP mRNAs. In addition to these findings, we find that a TSC2-null angiomyolipoma cell line forms vacuoles when treated with the proteasome inhibitor MG-132. We found these vacuoles to be derived from the ER and that rapamycin blocked their formation. Rapamycin also enhanced expansion of the ER during MG-132 stress and restored its degradation by autophagy. Taken together these findings suggest that bortezomib might be used to treat neoplastic growths associated with TSC. However, they also caution against combining specific cell death inducing agents with rapamycin during chemotherapy.

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