Externally disposed plasma membrane proteins. II. Metabolic fate of iodinated polypeptides of mouse L cells.

Abstract

The fate of the L-cell plasma membrane proteins labeled by enzymatic iodination was studied. The disappearance of label from growing cells exhibits a biphasic behavior, with 5-20% lost rapidly (t~ ~ 2 h) and 80-90% lost relatively slowly (t~ ~ 25-33 h). The loss is temperature dependent and serum independent, and is accompanied by the appearance of 51% [l~5l]monoiodotyrosine (MIT) in the medium by 47 h. A variable amount (1-14%) of acid-insoluble label can be recovered in the medium over 47 h. Sodium dodecyl sulfate (SDS)-polyacrylamide gel labeling patterns from cells cultured up to 48 h after iodination reveal no change in the relative distribution of radioactivity, indicating similar rates of degradation for most of the labeled membrane proteins.The fate of the labeled membrane proteins was studied at various times after phagocytosis of nondigestible polystyrene particles. Iodinated L cells phagocytose sufficient 1.1 /~m latex beads in 60 min to interiorize 15-30% of the total cell surface area. Electron microscope autoradiography confirmed that labeled membrane is internalized during phagocytosis. The latex-containing phagocytic vacuoles are isolated by flotation in a discontinuous sucrose gradient. 15-30% of the total incorporated label and a comparable percentage of alkaline phosphodiesterase I activity (PDase, a plasma membrane enzyme marker) are recovered in the phagocytic vacuole fraction. Lysosomal enzyme activities are found in the latex vacuole fraction, indicating formation of phagolysosomes. SDS gel analyses reveal that all of the radioactive proteins initially present on the intact cell's surface are interiorized to the same relative extent. Incorporated label and PDase activity disappear much more rapidly from the phagolysosomes than from the whole cell. In the phagolysosomal compartment, >70% of the TCA-precipitable labeled proteins and all of the PDase activity are lost rapidly (tv, = 1-2 h) but ~ 30% of the labeled proteins in this compartment are degraded with a 17-20-h half-life. The slowly degraded label is due to specific long-lived polypeptides, of 85,000 and 8,000--15,000 daltons, which remain in the phagolysosomal membrane up to 40 h after phagocytosis.

Zanvil Alexander Cohn 1926-1993.

“…In the latter, it became possible to document the relatively slow turnover of most plasma membrane proteins in cultured cells. When Hubbard and Cohn coaxed L cells to phagocytose latex particles, they found that the phagocytic vacuole membrane contained a representative sample of plasma membrane polypeptides, and that phagocytosis was associated with a much more rapid turnover of these polypeptides (103). Werb made similar findings on the effect of phagocytosis on the turnover of the plasma membrane enzyme, 5'-nucleotidase (79).…”

Role of autophagy, SQSTM1, SH3GLB1, and TRIM63 in the turnover of nicotinic acetylcholine receptors

“…In particular, the pathway leading to the degradation of this pentameric ion channel remains largely elusive. Evidence for lysosomal degradation of CHRN comes from studies using iodinated protein flux 45,46 and from the effects of lysosomal inhibitors on CHRN levels in C2C12 muscle cells. 18,47 Given the recent discussions about the physiological roles of TRIM63 in muscle atrophy and the findings of its possible function in the turnover of the CHRN, we focused on this process in more detail.…”

Kinetics of membrane internalization and recycling during pinocytosis in Dictyostelium discoideum.

“…During endocytosis, the plasma membrane invaginates and encloses extracellular fluid (pinocytosis) or particles (phagocytosis) in plasma-membrane-derived vesicles. This process leads to an extensive internalization of plasma membrane varying between 1 and 20 times the total cell surface area per hr, depending on cell type and culture conditions (6)(7)(8)(9)(10)(11)(12). However, during endocytosis no reduction of the cell surface area is observed.…”