The receptor recycling pathway contains two distinct populations of early endosomes with different sorting functions.

Receptor recycling involves two endosome populations, peripheral early endosomes and perinuclear recycling endosomes. In polarized epithelial cells, either or both populations must be able to sort apical from basolateral proteins, returning each to its appropriate plasma membrane domain. However, neither the roles of early versus recycling endosomes in polarity nor their relationship to each other has been quantitatively evaluated. Using a combined morphological, biochemical, and kinetic approach, we found these two endosome populations to represent physically and functionally distinct compartments. Early and recycling endosomes were resolved on Optiprep gradients and shown to be differentially associated with rab4, rab11, and transferrin receptor; rab4 was enriched on early endosomes and at least partially depleted from recycling endosomes, with the opposite being true for rab11 and transferrin receptor. The two populations were also pharmacologically distinct, with AlF4 selectively blocking export of transferrin receptor from recycling endosomes to the basolateral plasma membrane. We applied these observations to a detailed kinetic analysis of transferrin and dimeric IgA recycling and transcytosis. The data from these experiments permitted the construction of a testable, mathematical model which enabled a dissection of the roles of early and recycling endosomes in polarized receptor transport. Contrary to expectations, the majority (>65%) of recycling to the basolateral surface is likely to occur from early endosomes, but with relatively little sorting of apical from basolateral proteins. Instead, more complete segregation of basolateral receptors from receptors intended for transcytosis occurred upon delivery to recycling endosomes.

Isolation and characterization of the rat liver actin N-acetylaminopeptidase.

Actins from most eukaryotes undergo a unique post-translational modification of the amino terminus called "processing." Processing consists of the removal of an amino-terminal Ac-Met or Ac-Cys to leave an acidic amino-terminal residue. We have previously demonstrated that this reaction is not catalyzed by the ribosomally associated methionine aminopeptidase or by other previously described acetylaminopeptidases. Here we present the isolation and characterization of the actin N-acetylaminopeptidase (ANAP) from rat liver. A five-step purification protocol achieves a 4100-fold purification of the enzyme with an overall 8% recovery of activity. ANAP is a 77-kDa monomer with a pI of 4.6. Using unprocessed yeast actin as a substrate, the Km of ANAP is 3.5 microM. Purified ANAP was used to generate a polyclonal antibody. The antibody has been used along with activity assays to demonstrate the presence of ANAP in a variety of rat tissues. Finally, evidence is presented that in mammals, ANAP may function with a second, as yet unpurified, component to process actin amino termini.

Amino-terminal processing of actins mutagenized at the Cys-1 residue.

Most actins examined to date undergo a unique posttranslational modification termed processing, catalyzed by the actin N-acetylaminopeptidase. Processing is the removal of acetylmethionine from the amino terminus in class I actins with Met-Asp(Glu) amino termini. For class II actins with Met-X-Asp(Glu) amino termini, processing is the removal of the second residue as an N-acetylamino acid. Other cytosolic proteins with these amino termini are not processed suggesting that the reaction may be specific for actins. In actin, X is usually cysteine. However, there are some class II actins in which this residue is other than cysteine, suggesting a broader substrate specificity for actin N-acetylaminopeptidase than acetylmethionine or acetylcysteine. We constructed mutant actins in which this cysteine was replaced with serine, asparagine, glycine, aspartic acid, histidine, phenylalanine, and tyrosine and used these to determine the substrate specificity of rat liver actin N-acetylaminopeptidase in vitro. Amino-terminal acetylmethinonine was cleaved from adjacent aspartic acid, asparagine, or histidine, but not serine, glycine, phenylalanine, or tyrosine. Of the acetylated actin amino termini tested, only acetylmethionine and acetylcysteine were cleaved. Histidine was never N-acetylated and was not cleaved. When phenylalanine and tyrosine were adjacent to the initiator methionine, no initiator methionine was cleaved even though it was acetylated. These results suggest a narrow substrate specificity for the rat liver actin N-acetylaminopeptidase. They also demonstrate that the adjacent residue can effect actin N-acetylaminopeptidase specificity.

Unusual metabolism of the yeast actin amino terminus.

In this paper we have examined the post-translational modifications of the NH2 terminus of actin from the yeast Saccharomyces cerevisiae. Like actins examined previously, this actin contains an acetylated NH2 terminus. Actins in other organisms undergo a unique post-translational processing event in which the initial amino acid(s) are removed by an actin-specific processing enzyme in an acetylation-dependent reaction. This is defined as actin processing. In yeast, actin retains its initiator Met in vivo and is thus not processed even though a rat liver actin processing enzyme can process yeast actin in vitro. This lack of actin processing appears to be a general property of fungi, as the actin from three other species, Aspergillus nidulans, Schizosaccharomyces pombe, and Candida albicans are not NH2 terminally processed either. Yeast actin is a class I actin; its initiator Met directly precedes an acidic residue. We converted yeast actin to a class II species by inserting a Cys codon between the Met-1 and Asp-2 codons. In normal class II actins the Cys residue is removed as acetyl-Cys during processing. Neither the mutant actin nor chick beta-actin (a class I actin) are processed when expressed in yeast. S. cerevisiae thus appears to be also incapable of processing exogenous actins. Further study of the mutant actin containing a Cys at position 2 shows that 30-40% of this actin is stably unacetylated. This unacetylated actin does not have a shorter half-life than the acetylated form. From these studies we conclude that 1) NH2-terminal actin-specific processing is not required for actin function in yeast and three other fungi, 2) yeast are apparently incapable of processing any type of actin precursor, and 3) the stability of a yeast pseudo-class II actin is not affected by the acetylation state of the NH2 terminus.

Identification of N-acetylmethionine as the product released during the NH2-terminal processing of a pseudo-class I actin.

Genes for the various isoactins define two classes of actin. Class I actin genes code for Met-Asp(Glu)-actin, and class II actin genes code for Met-X-Asp(Glu)-actin where X is usually cysteine. Amino termini of both are removed in an acetylation-dependent processing reaction yielding acetyl-Asp(Glu)-actin. Both classes are processed at approximately equal rate (t1/2 = 15 min) in vivo. In vitro, class II actins are 90% processed by endogenous enzymes after 60 min in a rabbit reticulocyte lysate system, whereas class I actins are only minimally processed during this period. Using site-directed mutagenesis of a human skeletal muscle isoactin coupled with in vitro transcription and translation methods, we have synthesized a pseudo-class I actin in which the penultimate cysteine has been changed to an aspartic acid, thus placing a class I amino terminus on an otherwise class II actin molecule. The pseudo-class I actin was less than 20% processed during the translation period as determined by peptide mapping. It was further processed by exogenous processing enzyme at a rate compatible with a class I actin. These results indicate that the major actin determinant controlling differential actin-processing rates is the amino-terminal residue being cleaved, not the remaining structure of the actin molecule. We have also demonstrated for the first time that N-acetylmethionine is the immediately released product from the amino terminus of a pseudo-class I actin during processing.