Different ARF Domains Are Required for the Activation of Cholera Toxin and Phospholipase D

Gui-Feng Zhang,Walter A Patton,Fang-Jen S Lee,Marek Liyanage,Joong-Soo Han,Sue Goo Rhee,Joel Moss,Martha Vaughan

doi:10.1074/jbc.270.1.21

Abstract

ADP-ribosylation factors (ARFs), initially described as activators of cholera toxin ADP-ribosyltransferase activity, regulate intracellular vesicular membrane trafficking and stimulate a phospholipase D (PLD) isoform. ARF-like (ARL) proteins are structurally related to ARFs but do not activate cholera toxin and have relatively little effect on PLD. A new human ARL gene termed hARL1, which shares 57% amino acid identity with hARF1, was identified using a polymerase chain reaction-based cloning method. To determine whether different structural elements are responsible for the activation structural elements are responsible for the activation of the A subunit of cholera toxin and PLD, chimeric proteins were constructed by switching the amino-terminal 73 amino acids of ARF1 and ARL1. The recombinant rL73/F protein, in which the amino-terminal 73 amino acids of ARL1 replaced those of ARF1, activated the A subunit of cholera toxin, whereas the rF73/L protein, in which the NH2-terminal 73 amino acids of ARF1 replaced those of ARL1, was inactive. The two chimeric proteins had quite opposite effects on PLD activity. rF73/L activated PLD as effectively as rARF1, whereas rL73/F protein activated PLD only slightly. It appears that the amino-terminal region of ARF1 is not critical for its action as a GTP-dependent activator of cholera toxin, whereas it is necessary for activation of the putative effector enzyme, PLD.

Highlights

ADP-ribosylation factors (ARFs), initially described as activators of cholera toxin ADP-ribosyltransferase activity, regulate intracellular vesicular membrane trafficking and stimulate a phospholipase D (PLD) isoform
To determine whether different structural elements are responsible for the two ARF activities, chimeric proteins were constructed by switching the amino-terminal amino acid sequences of ARFI and ARLl
We report here the identification of different ARF domains involved in the activation of cholera toxin and phospholipase D

Summary

EXPERIMENTAL PROCEDURES

Materials-Enzymes for molecular biology were purchased from Boehringer Mannheim. ARF-stimulatable PLD was partially purified as described using heparin high performance liquid chromatography [18]. Sources of other materials have been published previously C18, 19). Isolation of Human ARLI eDNA-Human ARLI was isolated initially by polymerase chain reactions (PCR) from a yeast cDNAlibrary (Clontech)that was contaminated with human eDNA. Five PCRs were used to obtain segments of cDNAand to assemble a compositesequence of the full-length coding region. Primers 1222 C5'-TTGACACCAGACCAACTGGTAATG-3'), 1222B C5'-ACCGGCGCTCAGCTGGAATT-3'), 1218 C5'-GGTGGCGACGACTCCTGGAGCCCG-3'), and 1218A (5'CGTCAGTATCGGCGGAATTC-3') are complementary to sequences immediately upstream or downstream of the EcoRI insertion site of vector .\gtll. The nucleotide sequencers) reported in this paper has been submitted to the GenBankTM IEMBL Data Bank with accession number(s) L28997

11 Present address

RESULTS AND DISCUSSION

C Cardiolipin