Endocytosis allows cargo to enter a series of specialized endosomal compartments, beginning with early endosomes harboring Rab5 and its effector EEA1. There are, however, additional structures labeled by the Rab5 effector APPL1 whose role in endocytic transport remains unclear. It has been proposed that APPL1 vesicles are transport intermediates that convert into EEA1 endosomes. Here, we tested this model by analyzing the ultrastructural morphology, kinetics of cargo transport, and stability of the APPL1 compartment over time. We found that APPL1 resides on a tubulo-vesicular compartment that is capable of sorting cargo for recycling or degradation and that displays long lifetimes, all features typical of early endosomes. Fitting mathematical models to experimental data rules out maturation of APPL1 vesicles into EEA1 endosomes as a primary mechanism for cargo transport. Our data suggest instead that APPL1 endosomes represent a distinct population of Rab5-positive sorting endosomes, thus providing important insights into the compartmental organization of the early endocytic pathway.

Pharmacological modulators of host-microbial interactions can in principle be identified using highcontent screens. However, a severe limitation of this approach is the lack of insights into the mode of action of compounds selected during the primary screen. To overcome this problem, we developed a combined experimental and computational approach. We designed a quantitative multiparametric image-based assay to measure intracellular mycobacteria in primary human macrophages, screened a chemical library containing FDA-approved drugs, and validated three compounds for intracellular killing of M. tuberculosis. By integrating the multiparametric profiles of the chemicals with those of siRNAs from a genome-wide survey on endocytosis, we predicted and experimentally verified that two compounds modulate autophagy, whereas the third accelerates endosomal progression. Our findings demonstrate the value of integrating small molecules and genetic screens for identifying cellular mechanisms modulated by chemicals. Furthermore, selective pharmacological modulation of host trafficking pathways can be applied to intracellular pathogens beyond mycobacteria.

Retromer, a peripheral membrane protein complex, plays an instrumental role in host of cellular processes by its ability to recycle receptors from endosomes to the trans-Golgi network. It consists of two distinct sub-complexes, a membrane recognizing, sorting nexins (SNX) complex and a cargo recognition, vacuolar protein sorting (Vps) complex. Small

The flash-induced voltage response of halorhodopsin at high NaCl concentration comprises two main kinetic components. The first component with d dW1 W Ws does not exceed 4% of the overall response amplitude and is probably associated with the formation of the L (hR520) intermediate. The second main component with d dW1^2.5 ms which is independent of Cl 3 concentration can be ascribed to the transmembrane Cl 3 translocation during the L intermediate decay. The photoelectric response in the absence of Cl 3 has the opposite polarity and does not exceed 6% of the overall response amplitude at high NaCl concentration. A pH decrease results in substitution of the Cl 3

Laser flash-induced photovoltage responses of the D85S and D85T mutants as well as of the wild-type acid blue form are similar and reflect intraprotein charge redistribution caused by retinal isomerization. The Cl- -induced transition of all of these blue forms into purple ones is accompanied by the appearance of electrogenic stages, which is probably associated with Cl- translocation in the cytoplasmic direction. Cl- translocation efficiency of these purple forms is much lower than that of the proton transport by the wild-type bacteriorhodopsin. The values of the efficiency do not exceed 15, 8 and 3% for the D85T, D85S and wild-type acid purple form, respectively. Cl- induces an additional electrogenic phase in the photovoltage responses of the D85S mutant and the wild-type acid purple form. This phase is supposed to be associated with the reversible Cl- movement in the extracellular direction. It is interesting that this component is absent in the photovoltage response of the D85T mutant which has, like halorhodopsin, a threonine residue at position 85.

The intracellular localization of proteins to their specific compartments is a rich source of information for the study of biological processes. The colocalization of a protein with an established compartment marker is routinely measured from multi-color fluorescent microscopy images. Unfortunately, the apparent colocalization is a mixture of real and random colocalization. Random colocalization results from the limited resolution of the light microscope and the close location or occlusion of objects in the crowded cytoplasmic environment. Commonly used methods for the correction of random colocalization work well if the random colocalization is significantly smaller than the real colocalization. In the case where they have comparable values, the final result could be a senseless negative value. To solve this problem, we first developed a probabilistic model for the estimation of random colocalization and demonstrated that it produces results that coincide with the standard scramble method. Second, we developed a probabilistic model for the correction of random colocalization for the double and multiple colocalization of intracellular markers on vesicular structures. Our probabilistic method of estimation of real colocalization has two main advantages: 1) it never gives a negative colocalization value and 2) provides the estimation of colocalization uncertainty.

The scheme of the bacteriorhodopsin photocycle associated with a transmembrane proton transfer and electrogenesis is considered. The role of conformational changes in the polypeptide chain during the proton transport is discussed.

Electrogenic events in the E204Q bacteriorhodopsin mutant have been studied. A two-fold decrease in the magnitude of microsecond photovoltage generation coupled to M intermediate formation in the E204Q mutant is shown. This means that deprotonation of E204 is an electrogenic process and its electrogenicity is comparable to that of the proton transfer from the Schiff base to D85. pH dependence of the electrogenicity of M intermediate formation in the wild-type bacteriorhodopsin reveals only one component corresponding to the protonation of D85 in the bacteriorhodopsin ground state and transition of the purple neutral form into the blue acid form. Thus, the pK of E204 in the M state is close to the pK of D85 in the bacteriorhodopsin ground state ( 6 6 3) and far below the pK of the terminal proton release group (V6). It is concluded that E204 functions as the intermediate proton donor rather than the terminal proton release group in the bacteriorhodopsin proton pump.z 1998 Federation of European Biochemical Societies.