ISSN:0173-0835    

DOI:10.1002/elps.1150181403

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1997

数据来源: WILEY

摘要:

AbstractFollowing a concept developed by Bieret al. (Electrophoresis1993,14, 1011–1018), binary mixtures of amphoteric buffers with low conductivity and a good buffering capacity permit rapid rate zonal separation of proteins on a density gradient electrophoresis apparatus (7 cm, Ø 2.2 cm). At pH 8.66 and 250 V, β‐lactoglobulin (Mr36 600) was separated into the A and B isoforms within 44 min; human transferrin (Mr76 000–81 000) was separated into its sialylated glycoforms and carbonic anhydrase (Mr30 000) separated into its isoenzymes. From these results we arrive at the term high‐performance density gradient electrophoresis. Compartments belonging to the endosomal system were separated by density gradient electrophoresis. Early endosomes, recycling vesicles, intermediate endosomes, late endosomes and lysomes became well‐separated after 80 min at 10 mA using [125I]transferrin and horseradish peroxidase as reporter molecules in pulse‐chase regimes. Mixtures of Bier buffers and standard electrophoresis media permitted very short separation times (19 min at 10 mA) for the endosomal compartments. Concommittantly, endoplasmic reticulum and proteasomes were

ISSN:0173-0835    

DOI:10.1002/elps.1150181404

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1997

数据来源: WILEY

摘要:

AbstractThe separation of functional early and late endosomes from other cellular compartments by free‐flow electrophoresis (FFE) has been previously demonstrated in nonpolarized cells [1, 2]. Here, using125I‐labeled anti‐secretory component antibodies ([125I]SC Ab) and FITC‐labeled asialoorosomucoid (FITC‐ASOR) as markers of the transcytotic and lysosomal pathway, respectively, we demonstrate the separation of three distinct endosome subpopulations from polarized rat hepatocytes. Internalization of both markers at 16°C resulted in their accumulation in a common endosome compartment, indicating that both the transcytotic and the lysosomal pathways are arrested in the sorting early endosome at temperatures below 20°C. After chase of the markers from early endosomes into the transcytotic or the degradative route at 37°C, transcytotic endosomes carrying [125I]SC Ab migrated with an electrophoretic motility between early and late endosomes while late endosomes labeled with FITC‐ASOR were deflected more towards the anode than early endosomes. These data indicate that in rat hepatocytes, the transcytotic and lysosomal pathways utilize a common (i.e.early endosomes) and two distinct endosome subpopulations (i.e.transcytotic endosomes, late endosomes) prior to delivering proteins for biliary secretion or lysosomal degradation

ISSN:0173-0835    

DOI:10.1002/elps.1150181405

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1997

数据来源: WILEY

摘要:

AbstractPresentation of material derived from pathogenic organisms to the immune system requires uptake of antigens into antigen presenting cells, processing into peptide fragments and loading of the resulting fragments onto major histocompatibility complex (MHC) class II molecules. MHC class II‐restricted antigen presentation involves both the biosynthetic as well as the endocytic pathway of antigen‐presenting cells. In recent years, the general mechanisms that govern these processes have been delineated, and specialized organelles have been characterized in which processing and loading of antigens takes place. Here, we review the work that has led to the characterization of these MHC class II compartments, and describe the use of organelle electrophoresis and two‐dimensional gel electrophoresis to analyze the molecular composition of the different subcellular organelles involved in MHC class II‐restricted antigen presentation as well as in antigen

ISSN:0173-0835    

DOI:10.1002/elps.1150181406

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1997

数据来源: WILEY

摘要:

AbstractFree‐flow electrophoresis is a powerful tool to separate subcellular vesicles such as early and late endosomes from plasma membranes. Using this technique, the intracellular distribution of poliovirus type 2 Sabin (PV2) and its derived subviral particles was analyzed upon infection of HeLa cells. Comparison of various infection conditions showed that maximally 30% of total cell associated PV2 was found in endosomal compartments with the remainder being associated with plasma membrane fractions; 2% of viral label was recovered from the cytoplasm in form of free virions. Sucrose gradient centrifugation analysis of the viral material recovered from the respective fractions revealed that intracellular virus was exclusively in its native conformation. This is in sharp contrast to human rhinovirus serotype 2 (HRV2), which is rapidly modified to RNA‐free subviral particles upon accumulation in endosomes. The data suggest that productive poliovirus uncoating can occur at the plasma membrane whereas internalized virus is most probably abor

ISSN:0173-0835    

DOI:10.1002/elps.1150181407

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1997

数据来源: WILEY

摘要:

AbstractPolarized sorting of rhodopsin in retinal rod photoreceptors is mediated by post‐Golgi vesicles that bud from the trans‐Golgi network and fuse with the specialized domain of the plasma membrane in the rod inner segment. This domain surrounds the cilium that connects the inner segment and the rod outer segment to which mature rhodopsin is delivered. To dissect the sorting machinery that regulates budding, targeting, and fusion of rhodopsin carrier vesicles, their GTP‐binding protein composition has been studied using multiple means including high‐resolution two‐dimensional gel electrophoresis and [32P]GTP overlays of renatured proteins. These studies indicate a succession on rhodopsin‐bearing vesicles of rab6, rab11, rab3 and rab8, all members of the small GTP‐binding protein family of the known regulators of membrane trafficking. In this review the role of rab proteins in post‐Golgi trafficking of rhodops

ISSN:0173-0835    

DOI:10.1002/elps.1150181408

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1997

数据来源: WILEY

摘要:

AbstractOne of the most prominent features of pathogenic mycobacteria, which include the potent human pathogensMycobacterium tuberculosisandMycobacterium lepraeand their opportunistic relativesMycobacterium aviumandMycobacterium marinum, is their ability to survive and multiply in phagosomes of mononuclear phagocytic cells. The phagocytosed mycobacteria reside in a vacuolar compartment which is exempted from maturation into the phagolysosome. Recently, the arrest of the maturation of phagosomes containingM. tuberculosiscomplex organisms (Mycobacterium bovisBCG) has been linked to the accumulation on the phagosomal membrane of the small GTP binding protein rab5, specific for the control of fusion within the early endosomal compartment. Furthermore,M. bovisBCG phagosome is devoid of rab7, a rab protein associated with the late endosome. The selective accumulation of rab5 and exclusion of rab7 defines the check point that has been compromised in mycobacterial phagosome maturation. Here we summarize these observations and relates them to other phenomena in the area of membrane and protein trafficking with the emphasis on phagosomes containing intracellular pathogens.

ISSN:0173-0835    

DOI:10.1002/elps.1150181409

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1997

数据来源: WILEY

摘要:

AbstractThe endocytic compartment has emerged as a major regulator of the uptake and processing of circulating ligands, and has been extensively studied during the last decade. In this work, the polypeptides of the three endosomal fractions: compartment of uncoupling receptors and ligands (CURL), multivesicular bodies (MVB) and receptor recycling compartment (RRC), isolated from livers of estradiol‐treated rats, were analyzed by two‐dimensional gel electrophoresis. Silver‐stained gels revealed that although the three endosomal fractions shared a generally similar pattern of approximately 120 components, qualitative and quantitative differences between the three endocytic fractions could be demonstrated. The polypeptide composition of the bile was also studied and compared with ligands and proteins identified in the different endosomal fractions. One‐ and two‐dimensional gel electrophoresis and Western blotting were used to investigate the protein composition of the three isolated endocytic fractions and 39 proteins were identified. The distribution of identified receptors, ligands and structural proteins among the three endosomal fractions was in agreement with their expected functionalities and with the different endocytic pathways in the h

ISSN:0173-0835    

DOI:10.1002/elps.1150181410

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1997

数据来源: WILEY

摘要:

AbstractThe intramacrophage pathogenMycobacterium aviumresides in a vacuole which displays unusual fusion characteristics, expressed as both a failure to mature into phagolysosomes and a continued access to the early recycling pathway. In contrast, compartments containing inert IgG‐opsonized latex beads mature to phagolysosomes. Techniques were developed for the isolation of these particle‐containing phagosomes from macrophages to facilitate analysis of phagosomal constituents by electrophoresis and autoradiography. Metabolic labeling of macrophages followed by phagosome isolation and two‐dimensional polyacrylamide gel electrophoresis revealed only minor differences in the protein profiles between theM. aviumand IgG‐bead phagosomes despite the marked differences in the fusigenicity of the respective vacuoles. Pulse‐chase labeling experiments revealed greater differences in the accessibility ofMycobacterium aviumand IgG‐bead phagosomes to newly synthesized proteins. These phagosome isolation techniques were extended to analyze the protein synthesis profile of intracellularM. aviumfor comparison with bacteria that were metabolically labeled in broth culture. Not surprisingly, the majority of polypeptides in the bacilli were common to both growth conditions. However, despite these similarities, intracellularM. aviumexpress several unique proteins, most notably one abundant protein with a molecular weight of 51 kDa. In addition, the bacteria manifest a restricted set of proteins expressed while in stasis shortly afte

ISSN:0173-0835    

DOI:10.1002/elps.1150181411

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1997

数据来源: WILEY

摘要:

AbstractCells perform their multiple functions with the aid of a series of distinct membrane organelles. In the last years, many of these compartments have been isolated, purified, and extensively studied. The major roles of each organelle in the cell are well understood. However, most of the molecular basis by which they perform their functions is poorly known. The recent identification and study of a handful of proteins associated with endovacuolar compartments has had a major impact on the understanding of the molecular details of organelle functions even though two‐dimensional (2‐D) gel analysis indicates that hundreds of proteins are typically associated with a complex organelle. This shows that many details and surprises are still to come for cell biologists. In the present study, we have analyzed and compared different organelles of the endocytic and phagocytic apparatus using 2‐D gel electropho

ISSN:0173-0835    

DOI:10.1002/elps.1150181412

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1997

数据来源: WILEY