The polypeptides of three highly purified endosomal fractions isolated from the livers of oestradiol-treated rats were analysed by Western blotting, and the amount and distribution of intrinsic and cytoskeletal-associated proteins were quantified and studied. The 'late' endosomes [multivesicular bodies (MVBs)] had the lowest content of cytoskeletal-associated proteins, the most significant being the presence of 25% of the total dynein found in endosomes. The 'early' endosome [compartment of uncoupling receptors and ligands (CURL)] fraction contained kinesin (40% of the total in endosomes), dynein (23%), actin (15%) and tubulin (10%). The receptor-recycling compartment (RRC), also demonstrated to be involved in transcytosis, contained the largest number and enrichment of cytoskeletal proteins: actin (84% of the total in endosomes), alpha-actinin (90%), dynein (52%), tubulin (91%) and kinesin (45%). We also analysed and compared the presence of different endosomal markers such as Rab4, Rab5 and cellubrevin (vesicle soluble NSF attachment protein receptor) in CURL (41%, 15% and 60%) and in RRC (44%, 75% and 30% respectively). Finally, the expression of annexins I, II, IV and VI was studied: annexin I was equally distributed between MVBs and CURL; annexin II was highly enriched in RRC (95%), annexin IV was equally distributed between CURL and RRC, and annexin VI was enriched in CURL (57%). The results indicate that isolated rat liver endosomes contain all the required molecular machinery for the achievement of their role in intracellular trafficking.
Microbially-mediated methylation of arsenic (As) plays an important role in the As biogeochemical cycle, particularly in rice paddy soils where methylated As, generated microbially, is translocated into rice grains. The presence of the arsenite (As(III)) methyltransferase gene (arsM) in soil microbes has been used as an indication of their capacity for As methylation. Here, we evaluate the ability of seven microorganisms encoding active ArsM enzymes to methylate As. Amongst those, only the aerobic species were efficient methylators. The anaerobic microorganisms presented high resistance to As exposure, presumably through their efficient As(III) efflux, but methylated As poorly. The only exception were methanogens, for which efficient As methylation was seemingly an artifact of membrane disruption. Deletion of an efflux pump gene (acr3) in one of the anaerobes, Clostridium pasteurianum, rendered the strain sensitive to As and capable of more efficiently methylating As. Our results led to the following conclusions: (i) encoding a functional ArsM enzyme does not guarantee that a microorganism will actively drive As methylation in the presence of the metalloid and (ii) there is an inverse relationship between efficient microbial As efflux and its methylation, because the former prevents the intracellular accumulation of As.
1. The polypeptides of the three endosomal fractions isolated from livers of oestradiol-treated rats were analysed by two-dimensional gel electrophoresis. Silver-stained gels revealed that although the three endosomal fractions shared a generally similar pattern of approx. 120 components, qualitative and quantitative differences between the three endocytic fractions could be demonstrated. 2. The 'early' endosomes [compartment of uncoupling of receptors and ligands (CURL)] comprised the most complex fraction and contained most of the polypeptides found in the 'late' endosomes [multivesicular bodies (MVBs)] and the receptor recycling compartment (RRC). When CURL was analysed by two-dimensional gel electrophoresis after partition with Triton X-114, it showed the largest number of integral membrane polypeptides. 3. Some of the major receptors (polymeric immunoglobulin receptor, transferrin receptor, low-density lipoprotein receptor, asialoglycoprotein receptor, beta1-integrin, mannose 6-phosphate receptor, epidermal growth factor receptor and AGp110) and internalized ligands (IgA, IgG, albumin, haptoglobin, transferrin and alpha2-macroglobulin) were further studied by Western blotting. 4. The distribution of the identified receptors and ligands among the three endosomal fractions was in agreement with their expected functionalities. 5. The polypeptide composition of the bile was also examined and compared with ligands and proteins identified in the different endocytic fractions. 6. Finally, an electron microscopy study confirms the distinctive physical and ultrastructural features of the three isolated endosomal fractions.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.