scholarly journals Hydrolase secretion is a consequence of membrane recycling.

1984 ◽  
Vol 98 (1) ◽  
pp. 246-252 ◽  
Author(s):  
T C Hohman ◽  
B Bowers
Keyword(s):  
Plasma Membrane ◽  
Culture Medium ◽  
The Other ◽  
Cellular Proteins ◽  
Cellular Activity ◽  
Lysosomal Hydrolases ◽  
Ph Shift ◽  
Ph Dependent ◽  
Secondary Lysosomes ◽  
Energy Dependent

Acanthamoeba releases lysosomal hydrolases continuously into the culture medium. This release is specific for lysosomal hydrolases, but not other cellular proteins, and is energy dependent. The secreted hydrolases can be separated into two groups on the basis of their secretion kinetics: one is secreted at approximately 15% of the cellular activity per hour and the other at approximately 5%. Intracellularly the lysosomal hydrolases are restricted almost exclusively to secondary lysosomes where the hydrolases demonstrate a differential pH-dependent binding to membrane. Hydrolase secretion is not the result of secondary lysosomes' fusing with the plasma membrane since soluble and particulate lysosomal contents are not released at the same rate. Together the data suggest that the secreted hydrolases are trapped in shuttle vesicles that cycle membrane from secondary lysosomes to the cell surface. The inner membrane and content of these vesicles undergo a marked pH shift when, following fragmentation from lysosomes, these vesicles fuse with plasma membrane. This rapid pH shift and the differential pH-dependent membrane binding of hydrolases appear to account for the heterogeneous hydrolase secretion kinetics.

scholarly journals The membrane proteins of the vacuolar system. II. Bidirectional flow between secondary lysosomes and plasma membrane.

1980 ◽  
Vol 86 (1) ◽  
pp. 304-314 ◽  
Author(s):  
W A Muller ◽  
R M Steinman ◽  
Z A Cohn
Keyword(s):  
Plasma Membrane ◽  
Culture Medium ◽  
Fluid Phase ◽  
Membrane Flow ◽  
Em Autoradiography ◽  
Secondary Lysosomes ◽  
Bidirectional Flow ◽  
Vacuolar System ◽  
Representative Samples ◽  
Pinocytic Vesicles

Lactoperoxidase covalently coupled to latex spheres (LPO-latex) has been used to selectively iodinate the phagolysome (PL) membrane within living macrophages, as discussed in the accompanying article. This procedure labeled approximately 24 polypeptides in the PL membrane; these were similar to those iodinatable on the external surface of the plasma membrane (PM). We now report on the translocation and fate of these proteins when the cells are returned to culture. TCA-precipitable radioactivity was lost from cells with biphasic kinetics. 20-50% of the cell-associated radiolabel was rapidly digested (t 1/2 approximately equal to 1 h) and recovered in the culture medium as monoiodotyrosine. 50-80% of the label was lost slowly from cells ( 1/2 approximately equal to 24-30 h). Quantitative analysis of gel autoradiograms showed that all radiolabeled proteins were lost at the same rate in both the rapid and slow phases of digestion. Within 15-30 min aftr labeling of the PL membrane, EM autoradiography revealed that the majority of the cell-associated grains, which at time 0 were associated with PL, were now randomly dispersed over the plasmalemma. At this time, analysis of PM captured by a second phagocytic load revealed the presence of all labeled species originally present in the PL membrane. This demonstrated the rapid, synchronous centrifugal flow of PL polypeptides to the cell surface. Evidence was also obtained for the continuous influx of representative samples of the PM into the PL compartment by way of pinocytic vesicles. This was based on the constant flow of fluid phase markers into latex-containing PL and on the internalization of all iodinatable PM polypeptides into this locus. These observations provide evidence for the continuous, bidirectional flow of membrane polypeptides between the PM and the secondary lysosome and represent an example of a membrane flow and recycling mechanism.

scholarly journals A quantitative study of pinocytosis and intracellular proteolysis in rat peritoneal macrophages

1977 ◽  
Vol 168 (3) ◽  
pp. 365-372 ◽  
Author(s):  
M K Pratten ◽  
K E Williams ◽  
J B Lloyd
Keyword(s):  
Plasma Membrane ◽  
Binding Sites ◽  
Culture Medium ◽  
Peritoneal Macrophages ◽  
The Other ◽  
High Rate ◽  
Intracellular Proteolysis ◽  
Pinocytic Vesicles ◽  
Stimulation Of

A method for the culture of rat peritoneal macrophages in vitro is described, in which pinocytic uptake of colloidal [198 Au]gold, 125I--labelled poly(vinylpyrrolidone) and [14C]sucrose proceeds at contant and fairly reproducible rates for several hours. The rat of uptake of colloidal [198 Au]gold, which wxhibited some inter-batch variation, was approx. 100 times that of the other two substrates. Colloidal gold did not affect the rate of uptake of 125I-labelled poly(vinylpyrrolidone) and therefore its own high rate of uptake could not be attributed to a stimulation of the formation of pinocytic vesicles. It conclude that uptake of collodial gold is highly dependent on adsorption on binding sites on the plasma membrane. Uptake of formaldehyde-treated 125I-labelled bovine serum albumin was followed by the release of [125I]iodo-L-tyrosine into the culture medium and took place at a rate intermediate between those of collodial [198Au]gold and the other two non-digestible substrates, 125I-labelled poly(vinylpyrrolidone) and [14C]sucrose.

scholarly journals Calmodulin-microtubule association in cultured mammalian cells.

1984 ◽  
Vol 98 (3) ◽  
pp. 904-910 ◽  
Author(s):  
W J Deery ◽  
A R Means ◽  
B R Brinkley
Keyword(s):  
Plasma Membrane ◽  
Mammalian Cells ◽  
Cell System ◽  
The Other ◽  
Microtubule Assembly ◽  
Cytoplasmic Microtubule ◽  
Hypotonic Treatment ◽  
Cytoplasmic Microtubules ◽  
Triton X ◽  
Ca Sensitivity

A Triton X-100-lysed cell system has been used to identify calmodulin on the cytoskeleton of 3T3 and transformed SV3T3 cells. By indirect immunofluorescence, calmodulin was found to be associated with both the cytoplasmic microtubule complex and the centrosomes. A number of cytoplasmic microtubules more resistant to disassembly upon either cold (0-4 degrees C) or hypotonic treatment, as well as following dilution have been identified. Most of the stable microtubules appeared to be associated with the centrosome at one end and with the plasma membrane at the other end. These microtubules could be induced to depolymerize, however, by micromolar Ca++ concentrations. These data suggest that, by interacting directly with the microtubule, calmodulin may influence microtubule assembly and ensure the Ca++-sensitivity of both mitotic and cytoplasmic microtubules.

scholarly journals Effects of cytoplasmic acidification on clathrin lattice morphology.

1989 ◽  
Vol 108 (2) ◽  
pp. 401-411 ◽  
Author(s):  
J Heuser
Keyword(s):  
Plasma Membrane ◽  
Cultured Cells ◽  
Membrane Receptors ◽  
The Other ◽  
Culture Dish ◽  
Initial Curvature ◽  
Internal Ph ◽  
Cytoplasmic Acidification

Reducing the internal pH of cultured cells by several different protocols that block endocytosis is found to alter the structure of clathrin lattices on the inside of the plasma membrane. Lattices curve inward until they become almost spherical yet remain stubbornly attached to the membrane. Also, the lattices bloom empty "microcages" of clathrin around their edges. Correspondingly, broken-open cells bathed in acidified media demonstrate similar changes in clathrin lattices. Acidification accentuates the normal tendency of lattices to round up in vitro and also stimulates them to nucleate microcage formation from pure solutions of clathrin. On the other hand, several conditions that also inhibit endocytosis have been found to create, instead of unusually curved clathrin lattices with extraneous microcages, a preponderance of unusually flat lattices. These treatments include pH-"clamping" cells at neutrality with nigericin, swelling cells with hypotonic media, and sticking cells to the surface of a culture dish with soluble polylysine. Again, the unusually flat lattices in such cells display a tendency to round up and to nucleate clathrin microcage formation during subsequent in vitro acidification. This indicates that regardless of the initial curvature of clathrin lattices, they all display an ability to grow and increase their curvature in vitro, and this is enhanced by lowering ambient pH. Possibly, clathrin lattice growth and curvature in vivo may also be stimulated by a local drop in pH around clusters of membrane receptors.

Mechanism of cystine reaccumulation by cystinotic fibroblasts in vitro

1990 ◽  
Vol 10 (2) ◽  
pp. 225-229 ◽  
Author(s):  
Susan Forster ◽  
Lynne Scarlett ◽  
John B. Lloyd
Keyword(s):  
Plasma Membrane ◽  
Culture Medium ◽  
Human Fibroblasts ◽  
Competitive Inhibitor ◽  
Free Medium ◽  
Lysosome Membrane ◽  
Putative Mechanism

It is well established that when cystine-depleted cystinotic cells are cultured in cystine-containing medium, they reaccumulate cystine within their lysosomes more rapidly than when cultured in cystine-free medium. This has been a puzzling result, since the lysosome membrane of cystinotic cells is impermeable to cystine. To probe the mechanism of cystine reaccumulation, we have measured reaccumulation in the presence of colchicine, an inhibitor of pinocytosis, or of glutamate, a competitive inhibitor of cystine transport into human fibroblasts. Colchicine had no effect, thus eliminating pinocytosis as a putative mechanism for cystine translocation from the culture medium to the lysosomes. Glutamate, however, strongly inhibited cystine reaccumulation. It is concluded that the true mechanism is as follows. 1. Exogenous cystine crosses the plasma membrane on the cystine-glutamate porter. 2. Cystine is reduced in the cytoplasm by GSH. 3. The cysteine that is generated enters the lysosome, where it becomes cystine by participating in the reduction of cystine residues during intralysosomal proteolysis, or by autoxidation.

scholarly journals Role of the Endocytic Machinery in the Sorting of Lysosome-associated Membrane Proteins

2005 ◽  
Vol 16 (9) ◽  
pp. 4231-4242 ◽  
Author(s):  
Katy Janvier ◽  
Juan S. Bonifacino
Keyword(s):  
Plasma Membrane ◽  
Coat Protein ◽  
Membrane Proteins ◽  
Adaptor Protein ◽  
The Other ◽  
Sorting Signals ◽  
Efficient Delivery ◽  
Endocytic Machinery

The limiting membrane of the lysosome contains a group of transmembrane glycoproteins named lysosome-associated membrane proteins (Lamps). These proteins are targeted to lysosomes by virtue of tyrosine-based sorting signals in their cytosolic tails. Four adaptor protein (AP) complexes, AP-1, AP-2, AP-3, and AP-4, interact with such signals and are therefore candidates for mediating sorting of the Lamps to lysosomes. However, the role of these complexes and of the coat protein, clathrin, in sorting of the Lamps in vivo has either not been addressed or remains controversial. We have used RNA interference to show that AP-2 and clathrin—and to a lesser extent the other AP complexes—are required for efficient delivery of the Lamps to lysosomes. Because AP-2 is exclusively associated with plasma membrane clathrin coats, our observations imply that a significant population of Lamps traffic via the plasma membrane en route to lysosomes.

scholarly journals Lysosomal Hydrolase Mannose 6-Phosphate Uncovering Enzyme Resides in the trans-Golgi Network

2001 ◽  
Vol 12 (6) ◽  
pp. 1623-1631 ◽  
Author(s):  
Jack Rohrer ◽  
Rosalind Kornfeld
Keyword(s):  
Plasma Membrane ◽  
Fluorescent Protein ◽  
Lysosomal Hydrolases ◽  
Intracellular Location ◽  
Trans Golgi Network ◽  
Cytosolic Domain ◽  
Cytosolic Domains ◽  
Mannose 6 Phosphate ◽  
Green Fluorescent ◽  
Golgi Network

A crucial step in lysosomal biogenesis is catalyzed by “uncovering” enzyme (UCE), which removes a coveringN-acetylglucosamine from the mannose 6-phosphate (Man-6-P) recognition marker on lysosomal hydrolases. This study shows that UCE resides in the trans-Golgi network (TGN) and cycles between the TGN and plasma membrane. The cytosolic domain of UCE contains two potential endocytosis motifs: 488YHPL and C-terminal 511NPFKD. YHPL is shown to be the more potent of the two in retrieval of UCE from the plasma membrane. A green-fluorescent protein-UCE transmembrane-cytosolic domain fusion protein colocalizes with TGN 46, as does endogenous UCE in HeLa cells, showing that the transmembrane and cytosolic domains determine intracellular location. These data imply that the Man-6-P recognition marker is formed in the TGN, the compartment where Man-6-P receptors bind cargo and are packaged into clathrin-coated vesicles.

PI(4,5)P2 Clustering and Its Impact on Biological Functions

2021 ◽  
Vol 90 (1) ◽  
Author(s):  
Yi Wen ◽  
Volker M. Vogt ◽  
Gerald W. Feigenson
Keyword(s):  
Plasma Membrane ◽  
Cell Biology ◽  
Cellular Proteins ◽  
Annual Review ◽  
Publication Date ◽  
Biological Functions ◽  
Cellular Functions ◽  
Dynamic Distribution ◽  
Multivalent Cation ◽  
Lipid Environment

Located at the inner leaflet of the plasma membrane, phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2] comprises only 1–2 mol% of total PM lipids. With its synthesis and turnover both spatially and temporally regulated, PI(4,5)P2 recruits and interacts with hundreds of cellular proteins to support a broad spectrum of cellular functions. Several factors contribute to the versatile and dynamic distribution of PI(4,5)P2 in membranes. Physiological multivalent cations such as Ca2+ and Mg2+ can bridge between PI(4,5)P2 headgroups, forming nanoscopic PI(4,5)P2–cation clusters. The distinct lipid environment surrounding PI(4,5)P2 affects the degree of PI(4,5)P2 clustering. In addition, diverse cellular proteins interacting with PI(4,5)P2 can further regulate PI(4,5)P2 lateral distribution and accessibility. This review summarizes the current understanding of PI(4,5)P2 behavior in both cells and model membranes, with emphasis on both multivalent cation– and protein-induced PI(4,5)P2 clustering. Understanding the nature of spatially separated pools of PI(4,5)P2 is fundamental to cell biology. Expected final online publication date for the Annual Review of Biochemistry, Volume 90 is June 2021. Please see http://www.annualreviews.org/page/journal/pubdates for revised estimates.

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