Elucidation of the membrane topology of carnitine palmitoyltransferase (CPT) I showed that the extreme N-terminus is involved in determining the sensitivity of the liver (L) isoform to malonyl-CoA and suggested that interaction between the two cytosolic segments of the CPT I molecule determines the kinetic characteristics of the enzyme. Work with chimaeric liver/muscle-isoform (L/M) proteins constructed from all six possible combinations of three domains [N-terminus plus transmembrane domain 1 (TM1), loop plus TM2 and C-domain] expressed in Pichia pastoris showed that the precise N-C and TM1-TM2 pairings determine the overall kinetic parameters of the protein. Discrete short sequences within the respective N-terminal regions have negative or positive effects on malonyl-CoA sensitivity (L-isoform) or the K(m) for carnitine (M-isoform) in the full-length proteins, thus imparting to them their distinctive kinetic characteristics. Interactions within N-terminal domains also seem to be important in the targeting of the protein to microsomes in the P. pastoris expression system.
The submitochondrial distribution of the overt and latent carnitine palmitoyltransferases (CPT I and II respectively) of rat liver mitochondria were studied. Separation of outer and inner membranes, as well as of a fraction of intermediate density consisting of contact sites between the two membranes, was achieved, as judged by the distribution of marker enzymes. Both CPT I and CPT II were found to be enriched within the contact- site fraction of mitochondria. These data show that the two carnitine acyltransferases are distributed non-uniformly within their respective membranes, and that subpopulations of the two enzymes occur in close proximity within the mitochondrial membrane structure, while retaining their different accessibilities to cytosolic and matrix pools of metabolites. As the number of contact sites is known to vary with changes in the energy status of mitochondria, the possibility that such changes may acutely affect the proportion of CPT I within the distinctive lipid environment of the contact sites, and thus its overall kinetic characteristics, is discussed.
Abstract Cytokine-inducible SH2 domain–containing protein (CISH), a member of the suppressor of cytokine signaling family of negative feedback regulators, is induced by cytokines that activate STAT5 and can inhibit STAT5 signaling in vitro. However, demonstration of a definitive in vivo role for CISH during development has remained elusive. This study employed expression analysis and morpholino-mediated knockdown in zebrafish in concert with bioinformatics and biochemical approaches to investigate CISH function. Two zebrafish CISH paralogs were identified, cish.a and cish.b, with high overall conservation (43–46% identity) with their mammalian counterparts. The cish.a gene was maternally derived, with transcripts present throughout embryogenesis, and increasing at 4–5 d after fertilization, whereas cish.b expression commenced at 8 h after fertilization. Expression of cish.a was regulated by the JAK2/STAT5 pathway via conserved tetrameric STAT5 binding sites (TTCN3GAA) in its promoter. Injection of morpholinos targeting cish.a, but not cish.b or control morpholinos, resulted in enhanced embryonic erythropoiesis, myelopoiesis, and lymphopoiesis, including a 2- 3-fold increase in erythrocytic markers. This occurred concomitantly with increased activation of STAT5. This study indicates that CISH functions as a conserved in vivo target and regulator of STAT5 in the control of embryonic hematopoiesis.
The submitochondrial and subcellular distributions of the carnitine‐acylcarnitine translocase (CAC) have been studied. CAC is enriched to a much lesser extent than the carnitine palmitoyltransferases within the contact sites of mitochondria. A high‐abundance protein of identical molecular size as the mitochondrial CAC that is immunoreactive with an anti‐peptide antibody raised against a linear epitope of mitochondrial CAC is present in peroxisomes but not in microsomes. This suggests that CAC is targeted to at least two different locations within the liver cell and that acylcarnitine transport into peroxisomes is CAC mediated.
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