"The Membrane Polypeptides of the Vacuolar System: Composition and Recy" by William A. Muller
 

Student Theses and Dissertations

Date of Award

1981

Document Type

Thesis

Degree Name

Doctor of Philosophy (PhD)

Thesis Advisor

Zanvil Cohn

Keywords

phagolysosome, lactoperoxidase (LPO), macrophages, iodination, membrane recycling, polypeptides

Abstract

A method has been developed to deliver an iodinating system into the confines of the phagolysosome, allowing us to study the nature of the phagolysosomal membrane. Lactoperoxidase (LPO) is covalently coupled to carboxylated latex spheres (LPO-latex) in a stable enzymatically active form. The addition of LPO-Iatex to cultured macrophages leads to their rapid attachment, ingestion, and enclosure in a plasma membranederived phagocytic vacuole. These organelles rapidly fuse with preexisting lysosomes and are converted to phagolysosomes (PL) that demonstrate both acid phosphatase and lactoperoxidase activities. The exposure of LPO-Iatex containing cells to 125I- and an extracellular peroxide-generating system, glucose oxidase-glucose, at 4°C leads to incorporation of label into TCA-precipitable material. The incorporated cell-associated label was present as monoiodotyrosine; negligible amounts were found in lipids. Cell viability remained> 99%. Autoradiography at both the light and EM level revealed that > 97% of the cells were labeled, and quantitative analysis demonstrated the localization of grains to LPO-latex containing PL. PL were separated on sucrose gradients, and their radiolabel was confined almost exclusively to the membrane rather than soluble contents. SDS-polyacrylamide gel electrophoretic analysis of the peptides iodinated from within PL demonstrated at least 24 species with molecular weights ranging from 12,000 to 250,000. A very similar group of proteins was identified on the plasma membrane (PM) after surface iodination, and on latex phagosomes derived from iodinated PM. No novel proteins were detected in PL, either immediately after phagosome-lysosome fusion or after 1 h of intracytoplasmic residence. We conclude that the membrane proteins accessible to LPO-catalyzed iodination on the luminal surface of the PL and on the external face of the PM are similar, if not identical. When macrophages iodinated intralysosomally by LPO-latex were returned to culture at 37°, TCA-precipitable radioactivity was lost from cells with biphasic kinetics. 20-50% of the cell-associated radiolabel was rapidly digested (t1/2 ≅ 1 h) and recovered in the culture medium as monoiodotyrosine. 50-80% of the label was lost slowly from cells (t1/2 ≅ 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 after 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.

Comments

A thesis presented to the faculty of The Rockefeller University in partial fulfillment of the requirements for the degree of Doctor of Philosophy

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Creative Commons Attribution-NonCommercial-ShareAlike 4.0 International License
This work is licensed under a Creative Commons Attribution-NonCommercial-Share Alike 4.0 International License.

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