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Proteins targeted to the plasma membrane (PM) of cells are degraded at different rates. Sorting motifs contained within the cytoplasmic domains of transmembrane proteins, post-translational modifications (e.g. ubiquitination), and assembly into multiprotein or protein-lipid complexes all may affect the efficiency of endocytosis and recycling and influence the delivery to degradative compartments. Using the SNAP-tag labeling system, we examined the turnover of a model PM protein, the α chain of the interleukin-2 receptor (Tac). The surface lifetimes of SNAP-Tac fusions were influenced by their mode of entry into cells (clathrin-dependent versus clathrin-independent), their orientation in the PM (transmembrane versus glycosylphosphatidylinositol- anchored), and ubiquitination in their cytosolic domains. In addition, shedding of SNAP-Tac into the medium was greatly influenced by its O-linked glycosylation status. For a number of PM proteins, delivery to lysosomes and ectodomain shedding represent distinct parallel mechanisms to determine protein half-life. [ABSTRACT FROM AUTHOR]

Many plasma membrane (PM) proteins enter cells nonselectively through clathrin-independent endocytosis (CIE). Here, we present evidence that cytoplasmic sequences in three CIE cargo proteins--CD44, CD98, and CD147--were responsible for the rapid sorting of these proteins into endosomal tubules away from endosomes associated with early endosomal antigen 1 (EEA1). We found that Hook1, a microtubule- and cargo-tethering protein, recognized the cytoplasmic tail of CD147 to help sort it and CD98 into Rab22a-dependent tubules associated with recycling. Depletion of Hook1 from cells altered trafficking of CD44, CD98, and CD147 toward EEA1 compartments and impaired the recycling of CD98 back to the PM. In contrast, another CIE cargo protein, major histocompatibility complex class I, which normally traffics to EEA1 compartments, was not affected by depletion of Hook1. Loss of Hook1 also led to an inhibition of cell spreading, implicating a role for Hook1 sorting of specific CIE cargo proteins away from bulk membrane and back to the PM. [ABSTRACT FROM AUTHOR]

Describes the functions of the membrane protein kinesin as the motor for microtubule-mediated Golgi apparatus-to-endoplasmic reticulum (ER) traffic. Dependence of distribution and dynamics of both ER and Golgi complex on microtubules; Kinesin as the motor powering peripherally directed movements of membrane within the system; Structure-activity relationship of kinesin.

Details a study to uncover the mechanism by which Golgi membrane proteins are retained within the Golgi complex in the midst of a continuous flow of protein and lipid. Examination of the diffusional mobility of Golgi membrane components using fluorescence photobleaching recovery (FPR); What the diffusion coefficients were; Mobility and diffusion of the chimeric proteins; Conclusion about protein immobilization and Golgi targeting and retention.

The effects of thyroid hormone on nuclear-encoded mitochondrial inner membrane proteins were investigated by in vitro translation of the endogenous mRNA present in a postmitochondrial fraction from the livers of rats treated in vivo with hormone. The levels of the mRNAs were estimated by quantitative immunoabsorption of the translation mixture. Total protein synthesis was increased 2.6-fold after 4 days of in vivo hormone treatment, but only 10-15% of the polypeptides were dramatically altered (> 5-fold). Among the most highly elevated were cytochrome c1 (> 10-fold increase) and the Rieske iron-sulfur protein of the cytochrome bc1 complex. Other inner membrane proteins (core protein 1, β subunit of F1 ATPase, subunit IV of cytochrome oxidase, 3-hydroxybutyrate dehydrogenase) and non-mitochondrial proteins (rat serum albumin, β2-microglobulin) were not altered significantly by hormone treatment. Cytochrome c1 and the Rieske protein increased after 12 h of hormone treatment, a relatively early response in mammalian mitochondrial biogenesis. The possible significance of this response for the regulation of mitochondrial synthesis and assembly is discussed. [ABSTRACT FROM AUTHOR]

Several inner membrane proteins from rat liver mitochondria have been translated for the first time in rabbit reticulocyte lysates. These include the Rieske iron-sulfur protein, cytochrome c1 and core protein I of the cytochrome bc1 complex, the α and β subunits of F1 ATPase, and subunit IV of cytochrome oxidase. All were translated from free polysomes as larger-molecular-mass precursors, and were processed to their mature forms by isolated liver mitochondria or by the isolated mitochondrial matrix fraction. In vitro processing, catalyzed by the isolated matrix fraction, is inhibited by rhodamine 6G. The latter is a fluorescent probe, which accumulates specifically in mitochondria of whole cells and which is used extensively to visualize mitochondrial morphology. The concentration of rhodamine 6G required for inhibition in vitro is similar to that of o-phenanthroline. Rhodamine 6G inhibits matrix-catalyzed processing of all precursors tested, indicating that the mechanism of inhibition is common for a variety of functionally unrelated precursors. The novel action of rhodamine 6G reported here can form the basis for its inhibition of precursor processing in intact hepatoma cells.

Eukaryotic cells contain a variety of cytoplasmic Ca2+-dependent and Ca2+-independent phospholipase A2s (PLA2s; EC 2.3.1.2.3). However, the physiological roles for many of these ubiquitously-expressed enzymes is unclear or not known. Recently, pharmacological studies have suggested a role for Ca2+-independent PLA2 (iPLA2) enzymes in governing intracellular membrane trafficking events in general and regulating brefeldin A (BFA)-stimulated membrane tubulation and Golgi-to-endoplasmic reticulum (ER) retrograde membrane trafficking, in particular. Here, we extend these studies to show that membrane-permeant iPLA2 antagonists potently inhibit the normal, constitutive retrograde membrane trafficking from the trans-Golgi network (TGN), Golgi complex, and the ERGIC-53-positive ER-Golgi-intermediate compartment (ERGIC), which occurs in the absence of BFA. Taken together, these results suggest that iPLA2 enzymes play a general role in regulating, or directly mediating, multiple mammalian membrane trafficking events. [ABSTRACT FROM AUTHOR]