The topogenesis of membrane proteins with a single transmembrane (TM) segment is well understood. However, understanding the topogenesis and membrane assembly of membrane proteins with multiple TM segments (polytopic) is still incomplete. Recently, several studies on P-glycoprotein (Pgp) suggested that the topogenesis of polytopic membrane proteins is likely more complicated than anticipated. While studying the mechanism by which Pgp topogenesis is determined, we unexpectedly found that ribosomes or proteins associated with ribosomes are involved in regulating the membrane insertion and folding of Pgp during its translation. We discovered that when Pgp was translated by wheat germ ribosomes in vitro, TM3 could not reinitiate the insertion of the protein into microsomal membranes following the membrane insertion of TM1 and TM2. In contrast, TM3 could reinitiate membrane insertion when the protein was translated by rabbit reticulocyte ribosomes. These findings suggest that ribosomes or proteins associated with ribosomes play an important role in membrane insertion and folding of TM segments of Pgp and that rabbit reticulocyte and wheat germ ribosomes may use different mechanisms to control the membrane insertion of the same nascent peptide. We propose that ribosomes or proteins associated with ribosomes help reinitiate insertion of internal TM segments into the membrane by dissociation and reassociation with the protein-conducting channel in ER membranes.
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