Molecular Cloning, Characterization, and Dynamics of Rat Formiminotransferase Cyclodeaminase, a Golgi-associated 58-kDa Protein

Abstract

A peripherally associated 58-kDa Golgi protein (58K) of unknown function has been previously described (Bloom, G. S., and Brashear, T. A. (1989) J. Biol. Chem. 264, 16083-16092). To molecularly characterize 58K, we used a monoclonal anti-58K antibody (monoclonal antibody 58K-9) to screen a rat liver cDNA expression library. Positive clones were isolated, characterized, and partially sequenced. The obtained sequences show a high level of identity with sequences of porcine formiminotransferase cyclodeaminase (FTCD), suggesting that 58K is rat FTCD. Rat FTCD is structurally similar to porcine FTCD, a metabolic enzyme involved in conversion of histidine to glutamic acid, and exists in dimeric, tetrameric, and octameric complexes resistant to proteolysis. To define parameters of FTCD association with the Golgi, comparison of its behavior with various Golgi and ER-to-Golgi intermediate compartment marker proteins was examined under specific conditions. The results show that extraction parameters of FTCD are similar to those of GM130, a tightly associated Golgi matrix protein. FTCD appears to be a dynamic component of the Golgi, and a proportion of FTCD molecules cycle between the Golgi and earlier compartments of the secretory pathway. FTCD remains associated with Golgi fragments during microtubule disruption and is not released into cytosol during brefeldin A treatment. Instead, FTCD relocates from the Golgi, but the time course of its redistribution is distinct from that of mannosidase II relocation. FTCD is already dispersed into small punctate structures at a time when mannosidase II is still largely localized to Golgi structures. FTCD is not observed in tubules originating from the Golgi and containing mannosidase II. Instead, it appears to redistribute in small vesicles arranged in a linear "pearls on a string" pattern. These results suggest that FTCD relocation is temporally and spatially distinct from mannosidase II relocation and that FTCD provides a novel marker to study Golgi dynamics.

Microtubule actin crosslinking factor 1b: a novel plakin that localizes to the Golgi complex

“…Subsequently, the coverslips were washed with PBS and mounted onto slides with Aquamount (Lerner Laboratories) for indirect immunofluorescent microscopy. The following primary antibodies were used: rabbit polyclonal CU149 antibody, rabbit polyclonal CU119 antibody, rabbit polyclonal anti-HA antibody (BD Clontech), rabbit polyclonal anti-p130 antibody (Covance), mouse monoclonal anti-HA antibody (Covance), mouse monoclonal anti-GM130 antibody (BD transduction), mouse monoclonal anti-GOSR1 antibody (Abcam), monoclonal anti-formiminotransferase cyclodeaminase antibody [FTCD, also called p58; a gift from Elizabeth Sztul, University of Alabama at Birmingham, AL, USA; (Gao et al, 1998)], mouse monoclonal anti-␤-tubulin antibody (E7, culture supernatant), mouse monoclonal anti-GFP antibody (JL8; BD Biosciences) and mouse monoclonal anti-␤-actin antibody (AC15; Sigma). Each antibody was titred not to bleed through into the other channel.…”

IIGP, a member of the IFN inducible and microbial defense mediating 47 kDa GTPase family, interacts with the microtubule binding protein hook3

“…IIGP was detected in fractions II-IV, being most prominent in the fractions III and IV. The Golgi enriched fractions, namely fraction II and III, were identified with the anti-Golgi 58K mAb, detecting the Golgiassociated protein formiminotransferase cyclodeaminase (FTCD) (Bashour and Bloom, 1998;Gao et al, 1998). The ERenriched fractions IV and V were revealed by detection of the ER-resident membrane protein calnexin.…”

Distinct epitopes on formiminotransferase cyclodeaminase induce autoimmune liver cytosol antibody type 1

“…8 FTCD is a soluble cytosolic protein, but in cells appears to be preferentially associated with the cytosolic side of Golgi membranes. [9][10][11] In addition, FTCD appears to be a dynamic component of the Golgi, and cycles between the Golgi and earlier compartments of secretory pathways. 9 In this report, we show that a human liver cytosol protein immunoprecipitated with a pool of LC1-positive autoimmune sera is FTCD, and that LC1 sera recognize distinct epitopes on FTCD.…”

“…[9][10][11] In addition, FTCD appears to be a dynamic component of the Golgi, and cycles between the Golgi and earlier compartments of secretory pathways. 9 In this report, we show that a human liver cytosol protein immunoprecipitated with a pool of LC1-positive autoimmune sera is FTCD, and that LC1 sera recognize distinct epitopes on FTCD. We used full-length recombinant rat FTCD as antigenic substratum for immunodiffusion, immunoblotting, and immunoprecipitation experiments with a large series of LC1-positive and LC1-negative sera obtained from patients with type 2 autoimmune hepatitis.…”

“…Positive clones were isolated, characterized, and sequenced. 9 Sequences homologous to pig FTCD sequence were identified in GenBank using the BLAST program. The complete nucleotide sequence of rat FTCD is available at the accession number AF079233 from GeneBankTM/EMBL Data Bank.…”