In genetic screens for new endocytosis genes in Caenorhabditis elegans we identified RME-1, a member of a conserved class of Eps15-homology (EH)-domain proteins. Here we show that RME-1 is associated with the periphery of endocytic organelles, which is consistent with a direct role in endocytic transport. Endocytic defects in rme-1 mutants indicate that the protein is likely to have a function in endocytic recycling. Evidence from studies of mammalian RME-1 also points to a function for RME-1 in recycling, specifically in the exit of membrane proteins from recycling endosomes. These studies show a conserved function in endocytic recycling for the RME-1 family of EH proteins.

RME-1 is an Eps15-homology (EH)-domain protein that was identified in a genetic screen for endocytosis genes in Caenorhabditis elegans. When expressed in a CHO cell line, the worm RME-1 protein and a mouse homologue are both associated with the endocytic recycling compartment. Here we show that expression of a dominant-negative construct with a point mutation near the EH domain results in redistribution of the endocytic recycling compartment and slowing down of transferrin receptor recycling. The delivery of a TGN38 chimaeric protein to the trans-Golgi network is also slowed down. The function of Rme-1 in endocytic recycling is evolutionarily conserved in metazoans as shown by the protein's properties in C. elegans.

Although the distribution of the cation-independent mannose 6-phosphate receptor (CI-MPR) has been well studied, its intracellular itinerary and trafficking kinetics remain uncertain. In this report, we describe the endocytic trafficking and steady-state localization of a chimeric form of the CI-MPR containing the ecto-domain of the bovine CI-MPR and the murine transmembrane and cytoplasmic domains expressed in a CHO cell line. Detailed confocal microscopy analysis revealed that internalized chimeric CI-MPR overlaps almost completely with the endogenous CI-MPR but only partially with individual markers for the trans-Golgi network or other endosomal compartments. After endocytosis, the chimeric receptor first enters sorting endosomes, and it then accumulates in the endocytic recycling compartment. A large fraction of the receptors return to the plasma membrane, but some are delivered to the trans-Golgi network and/or late endosomes. Over the course of an hour, the endocytosed receptors achieve their steady-state distribution. Importantly, the receptor does not start to colocalize with late endosomal markers until after it has passed through the endocytic recycling compartment. In CHO cells, only a small fraction of the receptor is ever detected in endosomes bearing substrates destined for lysosomes (kinetically defined late endosomes). These data demonstrate that CI-MPR takes a complex route that involves multiple sorting steps in both early and late endosomes.

We examined the intracellular transport of sterol in living cells using a naturally fluorescent cholesterol analog, dehydroergosterol (DHE), which has been shown to mimic many of the properties of cholesterol. By using DHE loaded on methyl-␤-cyclodextrin, we followed this cholesterol analog in pulse-chase studies. At steady state, DHE co-localizes extensively with transferrin (Tf), a marker for the endocytic recycling compartment (ERC), and redistributes with Tf in cells with altered ERC morphology. Expression of a dominant-negative mutation of an ERC-associated protein, mRme-1 (G429R), results in the slowing of both DHE and Tf receptor return to the cell surface.

Hepatic secretion of apolipoprotein-B (apoB), the major protein of atherogenic lipoproteins, is regulated through posttranslational degradation. We reported a degradation pathway, post-ER presecretory proteolysis (PERPP), that is increased by reactive oxygen species (ROS) generated within hepatocytes from dietary polyunsaturated fatty acids (PUFA). We now report the molecular processes by which PUFA-derived ROS regulate PERPP of apoB. ApoB exits the ER; undergoes limited oxidant-dependent aggregation; and then, upon exit from the Golgi, becomes extensively oxidized and converted into large aggregates. The aggregates slowly degrade by an autophagic process. None of the oxidized, aggregated material leaves cells, thereby preventing export of apoBlipoproteins containing potentially toxic lipid peroxides. In summary, apoB secretory control via PERPP/autophagosomes is likely a key component of normal and pathologic regulation of plasma apoB levels, as well as a means for remarkably late-stage quality control of a secreted protein.

A significant fraction of internalized transferrin (Tf) concentrates in the endocytic recycling compartment (ERC), which is near the microtubule-organizing center in many cell types. Tf then recycles back to the cell surface. The mechanisms controlling the localization, morphology, and function of the ERC are not fully understood. We examined the relationship of Tf trafficking with microtubules (MTs), specifically the subset of stable, detyrosinated Glu MTs. We found some correlation between the level of stable Glu MTs and the distribution of the ERC; in cells with low levels of Glu MTs concentrated near to the centriole, the ERC was often tightly clustered, whereas in cells with higher levels of Glu MTs throughout the cell, the ERC was more dispersed. The clustered ERC in Chinese hamster ovary cells became dispersed when the level of Glu MTs was increased with taxol treatment. Furthermore, in a temperature-sensitive Chinese hamster ovary cell line (B104-5), the cells had more Glu MTs when the ERC became dispersed at elevated temperature. Microinjecting purified anti-Glu tubulin antibody into B104-5 cells at elevated temperature induced the redistribution of the ERC to a tight cluster. Microinjection of anti-Glu tubulin antibody slowed recycling of Tf to the cell surface without affecting Tf internalization or delivery to the ERC. Similar inhibition of Tf recycling was caused by microinjecting anti-kinesin antibody. These results suggest that stable Glu MTs and kinesin play a role in the organization of the ERC and in facilitating movement of vesicles from the ERC to the cell surface.

Abstract. Cytoplasmic dynein is a microtubulebinding protein which is considered to serve as a motor for retrograde organelle movement. In cultured fibroblasts, cytoplasmic dynein localizes primarily to lysosomes, membranous organelles whose movement and distribution in the cytoplasm have been shown to be dependent on the integrity of the microtubule cytoskeleton. We have recently identified conditions which lead to an apparent dissociation of dynein from lysosomes in vivo, indicating that alterations in membrane binding may be involved in the regulation of retrograde organelle movement (Lin, S. X. H., and C. A. Collins. 1993. J. Cell Sci. 105:579-588). Both brief serum withdrawal and low extracellular calcium levels induced this alteration, and the effect was reversed upon addition of serum or additional calcium. Here we demonstrate that the phosphorylation state of the dynein molecule is correlated with changes in its intracellular distribution in normal rat kidney fibroblasts. Dynein heavy chain phosphorylation level increased during serum starvation, and decreased back to control levels upon subsequent addition of serum. We found that okadaic acid, a phosphoprotein phosphatase inhibitor, mimicked the effects of serum starvation on both phosphorylation and the intracellular redistribution of dynein from a membrane-associated pool to one that was more soluble, with similar dose dependence for both phenomena. Cell fractionation by differential detergent extraction revealed that a higher proportion of dynein was present in a soluble pool after serum starvation than was found in comparable fractions from control cells. Our data indicate that cytoplasmic dynein is phosphorylated in vivo, and changes in phosphorylation state may be involved in a regulatory mechanism affecting the distribution of this protein among intracellular compartments.CUMULATING evidence indicates that retrograde and anterograde organelle transport along the microtubule network are mediated by the actions of microtubule-dependent motor molecules, cytoplasmic dynein, and kinesin (for review see Vallee and Shpetner, 1990;Walker and Sheetz, 1993). In contrast to the plethora of kinesin and kinesin-like molecules, only some of which may be anterograde organelle motors (for review see Endow, 1991;Goldstein, 1991;Walker and Sheetz, 1993), cytoplasmic dynein has been found in only one form in each cell type and species examined (Koonce et al., 1992;Paschal et al., 1992;Eshel et al., 1993;Li et al., 1993;Mikami et al., 1993;Zhang et al., 1993;Gibbons et al., 1994;Rasmusson et al., 1994). However, this retrograde translocator may carry out multiple cellular functions, as dynein has been localized to both membranous organdies and the mitotic apparatus in a number of cell types (Pfarr et al., 1990; Steuer et al., 1990;Lin and Collins, 1992 In our previous work cytoplasmic dynein in cultured fibroblasts was shown to be concentrated on large membrane-bounded organelles, identified as lysosomes (Lin and Collins, 1992). The association of dynein with these...

Novel molecular markers that are associated with prostate cancer (PCa) progression will provide valuable information in the diagnosis and treatment of the disease. Extracellular matrix metalloproteinase inducer (CD147) has been demonstrated to be involved in tumor invasion, metastasis, growth and survival. In our study, we examined whether the expression of CD147 can be used as a prognostic marker for predicting PCa progression. Tissue samples from 240 patients who received radical prostatectomy for PCa were obtained. CD147 expression in these samples was evaluated using immunohistochemical staining with a monoclonal antibody specifically against CD147. Increased expression of CD147 was correlated with higher Gleason scores (GS), positive surgical margin, prostate-specific antigen (PSA) failure, metastasis and reduced overall survival. Both univariate Cox regression analysis and multivariate analysis including competing biological variables demonstrated that increased CD147 expression was associated with increased risk for reduced PSA failure-free, metastasis-free and overall survival. Kaplan-Meier survival curves showed that the CD147 overexpression was a significant predictor for the PSA failurefree, metastasis-free and the overall survival in both pT2 and pT3 PCa patients. More significantly, higher expression of CD147 can serve as an independent prognostic predictor for PSA failure-free survival in PCa patients when they are stratified by GS. Our study results demonstrate the involvement of CD147 in PCa progression and suggest its potential role as an independent predictor of biochemical recurrence, development of metastasis and reduced overall survival in PCa.Prostate cancer (PCa) is one of the most common cancers and the second leading cause of cancer death in men. 1 Although patients with localized PCa can often be successfully treated with radical prostatectomy or radiation therapy, PCa deaths are usually the results of hormone refractory and metastatic disease. It is often difficult to identify patients who will progress, recur and require additional treatments. PCa prognosis varies significantly in patients according to clinical stage and pathological grade. More sensitive PCa novel molecular markers that are associated with biological aggressiveness and providing valuable information in the diagnosis and treatment of the disease are of particular importance. Currently, effective treatment of metastatic disease is one of the major therapeutic challenges in PCa treatment. 2 In recent years, many studies have focused on identifying nomograms to include various prognostic parameters to predict PCa outcome. [3][4][5] PCa cells with high-and low-metastatic potential vary in their biological properties, such as proliferation, adhesiveness, invasiveness and motility. These variations are the results of both germ line variation between individuals and somatic alterations of genes and gene expressions in cancer