ER-Bound Protein Tyrosine Phosphatase PTP1B Interacts with Src at the Plasma Membrane/Substrate Interface

PTP1B is an endoplasmic reticulum (ER) anchored enzyme whose access to substrates is partly dependent on the ER distribution and dynamics. One of these substrates, the protein tyrosine kinase Src, has been found in the cytosol, endosomes, and plasma membrane. Here we analyzed where PTP1B and Src physically interact in intact cells, by bimolecular fluorescence complementation (BiFC) in combination with temporal and high resolution microscopy. We also determined the structural basis of this interaction. We found that BiFC signal is displayed as puncta scattered throughout the ER network, a feature that was enhanced when the substrate trapping mutant PTP1B-D181A was used. Time-lapse and co-localization analyses revealed that BiFC puncta did not correspond to vesicular carriers; instead they localized at the tip of dynamic ER tubules. BiFC puncta were retained in ventral membrane preparations after cell unroofing and were also detected within the evanescent field of total internal reflection fluorescent microscopy (TIRFM) associated to the ventral membranes of whole cells. Furthermore, BiFC puncta often colocalized with dark spots seen by surface reflection interference contrast (SRIC). Removal of Src myristoylation and polybasic motifs abolished BiFC. In addition, PTP1B active site and negative regulatory tyrosine 529 on Src were primary determinants of BiFC occurrence, although the SH3 binding motif on PTP1B also played a role. Our results suggest that ER-bound PTP1B dynamically interacts with the negative regulatory site at the C-terminus of Src at random puncta in the plasma membrane/substrate interface, likely leading to Src activation and recruitment to adhesion complexes. We postulate that this functional ER/plasma membrane crosstalk could apply to a wide array of protein partners, opening an exciting field of research

A Comparison of FUV Auroral Emissions During the April 2002 Events as seen by the IMAGE/FUV and TIMED/GUVI Instruments

The auroral emissions that resulted from the series of solar particle events and magnetic storms during 14-24 April 2002 provide an excellent data set for the cross-comparison of the IMAGE/FUV and TIMED/GUVI auroral imagers. The IMAGE/FUV instrument comprises the SI spectral imager (121.8 nm and 135.6 nm) and the WIC imaging photometer (LBH) and observes the entire Earth from high Earth orbit. The TIMED/GUVI spectral imager (121.6 nm, 130.4 nm, 135.6 nm, LBH short, and LBH long) scans a nadir-to-limb swath from low Earth orbit. Although there is a large difference in spatial resolution, preliminary comparison of simultaneously-observed diffuse auroral emissions indicates fairly good agreement between the calibrated brightnesses determined for common spectral features. We will present a detailed simulation of one or more of the April 2002 events as seen by each imager to determine if a single description of the auroral precipitation can self-consistently account for the proton- and electron-generated FUV emissions observed from the two spacecraft

Intracellular Trafficking of the UL11 Tegument Protein of Herpes Simplex Virus Type 1

Growing evidence indicates that herpes simplex virus type 1 (HSV-1) acquires its final envelope in the trans-Golgi network (TGN). During the envelopment process, the viral nucleocapsid as well as the envelope and tegument proteins must arrive at this site in order to be incorporated into assembling virions. To gain a better understanding of how these proteins associate with cellular membranes and target to the correct compartment, we have been studying the intracellular trafficking properties of the small tegument protein encoded by the U(L)11 gene of HSV-1. This 96-amino-acid, myristylated protein accumulates on the cytoplasmic face of internal membranes, where it is thought to play a role in nucleocapsid envelopment and egress. When expressed in the absence of other HSV-1 proteins, the UL11 protein localizes to the Golgi apparatus, and previous deletion analyses have revealed that the membrane-trafficking information is contained within the first 49 amino acids. The goal of this study was to map the functional domains required for proper Golgi membrane localization. In addition to N-terminal myristylation, which allows for weak membrane binding, UL11 appears to be palmitylated on one or more of three consecutive N-terminal cysteines. Using membrane-pelleting experiments and confocal microscopy, we show that palmitylation of UL11 is required for both Golgi targeting specificity and strong membrane binding. Furthermore, we found that a conserved acidic cluster within the first half of UL11 is required for the recycling of this tegument protein from the plasma membrane to the Golgi apparatus. Taken together, our results demonstrate that UL11 has highly dynamic membrane-trafficking properties, which suggests that it may play multiple roles on the plasma membrane as well as on the nuclear and TGN membranes