Subcellular localization of the sulphation reaction of heparan sulphate synthesis and transport of the proteoglycan to the cell surface in rat liver

Graham, John M.; Winterbourne, David J.

doi:10.1042/bj2520437

Cited by 17 publications

(18 citation statements)

References 42 publications

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“…Here, we have identified and partially characterized a post-TGN vesicular fraction carrying HSPG in rat hepatocytes. As shown previously (Graham and Winterbourne, 1988), in rat hepatocytes, HSPG is transported exclusively to the plasma membrane and not to endosomal/lysosomal compartments. Therefore, the HSPG-transporting post-TGN vesicles described here are constitutive secretory vesicles.…”

Section: Characterization Of the Hspg-carrying Post-tgn Vesiclessupporting

confidence: 76%

“…Aliquots (~100 td) of the fractions obtained from the sucrose-and Nycodenz gradients were assayed by precipitation of macromolocules using cetylpyridinium chloride (Graham and Winterbourne, 1988).…”

Section: Quantification Of 35s-labeled Macromoleculesmentioning

confidence: 99%

“…After 12 and 30 min of chase, an increasing portion of 35S-labeled macromolecules had returned to the bottom of the gradient. The transport of HSPGs from the TGN to the cell surface does not involve the endocytic compartments (Graham and Winterbourne, 1988). Therefore, the fraction of labeled macromolecules appear~ in the bosom fractions of the gradient after 12 and 30 min of chase represents HSPGs, which had reached the cell surface.…”

Section: Identification Of Secretory Vesicles Carrying ~S-sulfated Mamentioning

confidence: 99%

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ADP ribosylation factor and a 14-kD polypeptide are associated with heparan sulfate-carrying post-trans-Golgi network secretory vesicles in rat hepatocytes.

Nickel¹,

Huber²,

Kahn³

et al. 1994

The Journal of Cell Biology

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Abstract. Constitutive secretory vesicles carrying heparan sulfate proteoglycan (HSPG) were identified in isolated rat hepatocytes by pulse-chase experiments with psS]sulfate and purified by velocity-controlled sucrose gradient centrifugation followed by equilibrium density centrifugation in Nycodenz. Using this procedure, the vesicles were separated from plasma membranes, Golgi, trans-Golgi network (TGN-), ER, endosomes, lysosomes, transcytotic vesicles, and mitochondria. The diameter of these vesicles was ,,o100-200 nm as determined by electron microscopy. A typical coat structure as described for intra-Golgi transport vesicles or clathrin-coated vesicles could not be seen, and the vesicles were not associated with the coat protein B-COP. Furthermore, the vesicles appear to represent a low density compartment (1.05-1.06 g/ml).Other constitutively secreted proteins (rat serum albumin, apolipoprotein E, and fibrinogen) could not be detected in purified HSPG-carrying vesicles, but banded in the denser fractions of the Nycodenz gradient. Moreover, during pulse-chase labeling with [35S]methionine, labeled albumin did not appear in the post-TGN vesicle fraction carrying HSPGs. These findings indicate sorting of HSPGs and albumin into different types of constitutive secretory vesicles in hepatocytes. Two proteins were found to be tightly associated with the membranes of the HSPG carrying vesicles: a member of the ADP ribosylatlon factor family of small guanine nucleotide-binding proteins and an unknown 14-kD peripheral membrane protein (VAPP14).Concerning the secretory pathway, we conclude from these results that ADP ribosylation factor proteins are not only involved in vesicular transport from the ER via the Golgi to the TGN, but also in vesicular transport from the TGN to the plasma membrane.

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Section: Characterization Of the Hspg-carrying Post-tgn Vesiclessupporting

confidence: 76%

Section: Quantification Of 35s-labeled Macromoleculesmentioning

confidence: 99%

Section: Identification Of Secretory Vesicles Carrying ~S-sulfated Mamentioning

confidence: 99%

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ADP ribosylation factor and a 14-kD polypeptide are associated with heparan sulfate-carrying post-trans-Golgi network secretory vesicles in rat hepatocytes.

Nickel¹,

Huber²,

Kahn³

et al. 1994

The Journal of Cell Biology

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“…Subsequently, we have studied the constitutive TG/TGN to cell surface membrane transport step. Thus, [ 35 S]sulfate‐labelled GAGs were used as markers [32,35] because GAGs are soluble and are sulfated at the most distal Golgi compartments [36,37]. As can be observed in Fig.…”

Section: Resultsmentioning

confidence: 99%

Morphological and biochemical analysis of the secretory pathway in melanoma cells with distinct metastatic potential

et al. 1999

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In this report, we have investigated whether alterations of the morphological and functional aspects of the biosecretory membrane system are associated with the metastatic potential of tumor cells. To this end, we have analyzed the morphology of the Golgi complex, the cytoskeleton organization and membrane trafficking steps of the secretory pathway in two human melanoma A375 cell line variants with low (A375-P) and high metastatic (A375-MM) potential. Immunofluorescence analysis showed that in A375-P cells, the Golgi complex showed a collapsed morphology. Conversely, in A375-MM cells, the Golgi complex presented a reticular and extended morphology. At the ultrastructural level, the Golgi complex of A375-P cells was fragmented and cisternae were swollen. When the cytoskeleton was analyzed, the microtubular network appeared normal in both cell variants, whereas actin stress fibers were largely absent in A375-P, but not in A375-MM cells. In addition, the F-actin content in A375-P cells was significantly lower than in A375-MM cells. These morphological differences in A375-P cells were accompanied by acceleration and an increase in the endoplasmic reticulum to Golgi and the trans-Golgi network to cell surface membrane transport, respectively. Our results indicate that in human A375 melanoma cells, metastatic potential correlates with a well-structured morphofunctional organization of the Golgi complex and actin cytoskeleton.z 1999 Federation of European Biochemical Societies.

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“…At various time points, cells and medium were separated by centrifugation at 350 ϫ g for 5 min. Protein-bound radioactivity was determined by scintillation counting after precipitation with cetylpyridinium chloride (44).…”

Section: Methodsmentioning

confidence: 99%

Sorting of Lysosomal Membrane Glycoproteins lamp-1 and lamp-2 into Vesicles Distinct from Mannose 6-Phosphate Receptor/γ-Adaptin Vesicles at the trans-Golgi Network

Karlsson

Carlsson

1998

Journal of Biological Chemistry

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Newly synthesized lysosomal membrane glycoproteins lamp-1 and lamp-2 are primarily sorted at the trans-Golgi network (TGN) by recognition of a tyrosinebased signal sequence in their cytoplasmic tails. It is presently unclear how this signal is recognized and what type of vesicle transports lamp-1 and lamp-2. Here, we describe a method to generate transport vesicles containing lamp proteins from the TGN in vitro. The method is based on incorporation of radioactive sialic acid in glycoproteins at the TGN by incubation of membranes with tritiated CMP-sialic acid. The generation of vesicles from labeled membranes required ATP and cytosol, and was temperature-dependent and brefeldin Asensitive. Analysis on Nycodenz gradients revealed that lamp-vesicles were distinct from vesicles containing ␥-adaptin and mannose 6-phosphate receptor (MPR). Moreover, both these types of vesicles migrated differently than vesicles containing proteins destined for the plasma membrane. The conclusion that lamps and MPRs are sorted into different vesicles was further strengthened by the finding that whereas wortmannin both in vitro and in vivo inhibited the production of ␥-adaptin/ MPR-containing vesicles, this drug had no effect on the generation of lamp-vesicles and on the sorting of lamps. The results indicate that membrane proteins containing tyrosine-based motifs for sorting at the TGN are segregated from clathrin-coated vesicles containing MPRs.

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Subcellular localization of the sulphation reaction of heparan sulphate synthesis and transport of the proteoglycan to the cell surface in rat liver

Cited by 17 publications

References 42 publications

ADP ribosylation factor and a 14-kD polypeptide are associated with heparan sulfate-carrying post-trans-Golgi network secretory vesicles in rat hepatocytes.

ADP ribosylation factor and a 14-kD polypeptide are associated with heparan sulfate-carrying post-trans-Golgi network secretory vesicles in rat hepatocytes.

Morphological and biochemical analysis of the secretory pathway in melanoma cells with distinct metastatic potential

Sorting of Lysosomal Membrane Glycoproteins lamp-1 and lamp-2 into Vesicles Distinct from Mannose 6-Phosphate Receptor/γ-Adaptin Vesicles at the trans-Golgi Network

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