Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:2.3.1.28 (chloramphenicol acetyltransferase)
 5,100 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The purpose of this research was twofold, a) to directly demonstrate import in vivo of a native plant peroxisomal protein into peroxisomes of transiently transfected mammalian cells, and b) to identify the targeting signal and amino acid substitutions thereof which preserve translocation of this plant protein into these peroxisomes. The protein selected for study was cottonseed isocitrate lyase (ICL), a glyoxylate cycle enzyme which participates in storage oil mobilization in oilseed cotyledons. Cultured mammalian cells were selected as the import system because of previous success by others with transient transfections and import of heterologous (not plant, however) proteins, and because neither a plant in vitro or transient in vivo import system was established. Optimized transient transfections of cultured CV-1 monkey kidney, mouse L, HeLa, and CHO cells resulted in punctate, anticottonseed-ICL-dependent immunofluorescent patterns. Colocalization in a CVH Px110 cell line of ICL with either endogenous catalase or with stably expressed CAT-PMP20/AKL (chloramphenicol acetyltransferase with a C-terminal-appended 12 amino acids ending with Ala-Lys-Leu) demonstrated targeting of ICL to peroxisomes. Direct evidence for translocation of ICL into CHO cell peroxisomes was obtained from digitonin permeabilization experiments. The necessity of the C-terminal tetrapeptide, KARM-COOH, was demonstrated in CHO and CV-1 cells when removal of this tetrapetide (leaving ICL-VVA-COOH) abolished import into peroxisomes. This result is in general agreement with Olsen et al. (The Plant Cell 5, 941-952 (1993)) who demonstrated that the 37 C-terminal amino acids of oilseed rape ICL were necessary for import in vivo in transgenic plants. The findings of Behari and Baker (J. Biol. Chem. 268, 7315-7322 (1993)), however, indicate that the C-terminal portion of castor bean ICL is dispensible for import in vitro. Single or multiple conservative amino acid substitutions at each position of the C-terminal tripeptide of native cottonseed ICL (S for A, K for R, L for M, SK for AR, SKL for ARM) preserved import of the enzyme in vivo into CHO cell peroxisomes. The demonstrated targeting and translocation of plant ICL and C-terminal modifications thereof into mammalian cell peroxisomes provide important additional evidence for evolutionary conservation of peroxisome import machinery, especially relative to the PTS1 sequence.
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PMID:Conservative amino acid substitutions of the C-terminal tripeptide (Ala-Arg-Met) on cottonseed isocitrate lyase preserve import in vivo into mammalian cell peroxisomes. 772 Jul 22

The function of the C-terminal tripeptide targeting signal responsible for microbody targeting in many eukaryotes has been investigated in the filamentous fungus Neurospora crassa. Using an in-vivo targeting assay that employs transformants carrying C-terminally-modified versions of the bacterial enzyme chloramphenicol acetyltransferase (CAT), it has been demonstrated that C-terminal tripeptide-dependent import occurs most efficiently in response to nutritional acetate-induction. Under these conditions Neurospora generates a specialized organelle, the glyoxysome, which carries the enzymes responsible for the glyoxylate cycle and can be distinguished from peroxisome-like microbodies that contain catalase. Moreover, several C-terminal peptides have been tested in this system to extend the tripeptide targeting consensus to A/C/G/S-H/K/Q/R-I/L/V. However, the tripeptide analogue, ARM, found at the C-terminus of the glyoxylate cycle enzyme isocitrate lyase in higher plants, does not apparently function here.
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PMID:The function and specificity of the C-terminal tripeptide glyoxysomal targeting signal in Neurospora crassa. 787 36

Isocitrate lyase (IL) is an essential enzyme in the glyoxylate cycle, which is a pathway involved in the mobilization of stored lipids during postgerminative growth of oil-rich seedlings. We determined experimentally the necessary and sufficient peroxisome targeting signals (PTSs) for cottonseed, oilseed rape, and castor bean ILs in a well-characterized in vivo import system, namely, suspension-cultured tobacco (Bright Yellow) BY-2 cells. Results were obtained by comparing immunofluorescence localizations of wild-type and C-terminal-truncated proteins transiently expressed from cDNAs introduced by microprojectile bombardment. The tripeptides ARM-COOH (on cottonseed and castor bean ILs) and SRM-COOH (on oilseed rape IL) were necessary for targeting and actual import of these ILs into glyoxysomes, and ARM-COOH was sufficient for redirecting chloramphenicol acetyltransferase (CAT) from the cytosol into the glyoxysomes. Surprisingly, IL and CAT subunits without these tripeptides were still acquired by glyoxysomes, but only when wild-type IL or CAT-SKL subunits, respectively, were simultaneously expressed in the cells. These results reveal that targeting signal-depleted subunits are being piggybacked as multimers to glyoxysomes by association with subunits possessing a PTS1. Targeted multimers are then translocated through membrane pores or channels to the matrix as oligomers or as subunits before reoligomerization in the matrix.
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PMID:Oilseed isocitrate lyases lacking their essential type 1 peroxisomal targeting signal are piggybacked to glyoxysomes. 906 50