Interaction of the Deubiquitinating Enzyme Ubp2 and the E3 Ligase Rsp5 Is Required for Transporter/Receptor Sorting in the Multivesicular Body Pathway

Protein ubiquitination is essential for many events linked to intracellular protein trafficking. We sought to elucidate the possible involvement of the S. cerevisiae deubiquitinating enzyme Ubp2 in transporter and receptor trafficking after we (this study) and others established that affinity purified Ubp2 interacts stably with the E3 ubiquitin ligase Rsp5 and the (ubiquitin associated) UBA domain containing protein Rup1. UBP2 interacts genetically with RSP5, while Rup1 facilitates the tethering of Ubp2 to Rsp5 via a PPPSY motif. Using the uracil permease Fur4 as a model reporter system, we establish a role for Ubp2 in membrane protein turnover. Similar to hypomorphic rsp5 alleles, cells deleted for UBP2 exhibited a temporal stabilization of Fur4 at the plasma membrane, indicative of perturbed protein trafficking. This defect was ubiquitin dependent, as a Fur4 N-terminal ubiquitin fusion construct bypassed the block and restored sorting in the mutant. Moreover, the defect was absent in conditions where recycling was absent, implicating Ubp2 in sorting at the multivesicular body. Taken together, our data suggest a previously overlooked role for Ubp2 as a positive regulator of Rsp5-mediated membrane protein trafficking subsequent to endocytosis

Federal Building and Fire Safety Investigation of the World Trade Center Disaster: Occupant Behavior, Egress, and Emergency Communications

Peer reviewed: NoNRC publication: Ye

Movement on Stairs During Building Evacuations

The time that it takes an occupant population to reach safety when descending a stair during building evacuations is typically estimated by measureable engineering variables such as stair geometry, speed, stair density, and pre-observation delay. In turn, engineering models of building evacuation use these variables to predict the performance of egress systems for building design, emergency planning, or event reconstruction. As part of a program to better understand occupant movement and behavior during building emergencies, the Engineering Laboratory at the National Institute of Standards and Technology (NIST) has been collecting stair movement data during fire drill evacuations of office and residential buildings. These data collections are intended to provide a better understanding of this principal building egress feature and develop a technical foundation for future codes and standards requirements. NIST has collected fire drill evacuation data in 14 buildings (11 office buildings and 3 residential buildings) ranging from six to 62 stories in height that have included a range of stair widths and occupant densities. A total of more than 22000 individual measurements are included in the data set. This report provides details of the data collected, an analysis of the data, and examples of the use of the data. The intention is to better understand movement during stair evacuations and provide data to test the predictive capability of building egress models. While mean movement speeds in the current study of 0.44 m/s ± 0.19 m/s are observed to be quite similar to the range of values in previous studies, mean local movement speeds as occupants traverse down the stairs are seen to vary widely within a given stair, ranging from 0.10 m/s ± 0.008 m/s to 1.7 m/s ± 0.13 m/s. These data provide confirmation of the adequacy of existing literature values typically used for occupant movement speeds and provide updated data for use in egress modeling or other engineering calculations

Pheromone-induced anisotropy in yeast plasma membrane phosphatidylinositol-4,5-bisphosphate distribution is required for MAPK signaling

During response of budding yeast to peptide mating pheromone, the cell becomes markedly polarized and MAPK scaffold protein Ste5 localizes to the resulting projection (shmoo tip). We demonstrated before that this recruitment is essential for sustained MAPK signaling and requires interaction of a pleckstrin homology (PH) domain in Ste5 with phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2] in the plasma membrane. Using fluorescently tagged high-affinity probes specific for PtdIns(4,5)P2, we have now found that this phosphoinositide is highly concentrated at the shmoo tip in cells responding to pheromone. Maintenance of this strikingly anisotropic distribution of PtdIns(4,5)P2, stable tethering of Ste5 at the shmoo tip, downstream MAPK activation, and expression of a mating pathway-specific reporter gene all require continuous function of the plasma membrane-associated PtdIns 4-kinase Stt4 and the plasma membrane-associated PtdIns4P 5-kinase Mss4 (but not the Golgi-associated PtdIns 4-kinase Pik1). Our observations demonstrate that PtdIns(4,5)P2 is the primary determinant for restricting localization of Ste5 within the plasma membrane and provide direct evidence that an extracellular stimulus-evoked self-reinforcing mechanism generates a spatially enriched pool of PtdIns(4,5)P2 necessary for the membrane anchoring and function of a signaling complex