Identification of a Manduca sexta NSF ortholog, a member of the AAA family of ATPases.
Transport between intracellular compartments requires the activity of an N-ethylmaleimide-sensitive fusion protein (NSF). NSF is a member of a growing family of ATPases regulating several membrane fusion reactions. We have cloned the NSF ortholog from the moth, Manduca sexta (MsNSF). MsNSF is highly conserved in domains critical for NSF function in vertebrates. MsNSF codes for a protein of 745 amino acids, translating to a M(r) of 83kDa in vitro. MsNSF is 72% and 61% similar in amino acid sequence to Drosophila and vertebrate NSFs, respectively. We expressed the D1 ATP domain of MsNSF toward which antibodies selective to MsNSF were generated. Affinity purified alpha-MsNSF antibodies detect a 83kDa protein which is highly enriched in nervous tissues. Levels of MsNSF expression are substantially lower in other tissues examined. Anti-MsNSF antibodies are capable of inhibiting vertebrate intra-Golgi transport of a cargo protein in vitro. The identification of NSF ortholog from Manduca, whose neuroendocrine system is well studied, should facilitate isolation of complexes involved in protein trafficking from insect models. Phylogenetic analysis of NSF and related proteins suggests that the members of the AAA family arose from different ancestors, since the ingroup was not monophyletic. Proteasomal subunits and p97 homologs form two distinct subfamilies, while NSF homologs branch in to the third.
Pullikuth
A K
AK
Department of Neuroscience, University of California, Riverside, CA, USA.
Gill
S S
SS
D000251:Adenosine Triphosphatases / Q000502:physiology*; D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology / Q000494:pharmacology; D000915:Antibody Affinity; D000918:Antibody Specificity; D001483:Base Sequence; D001692:Biological Transport / Q000187:drug effects; D016466:CHO Cells; D002352:Carrier Proteins / Q000235:genetics* / Q000276:immunology / Q000502:physiology; D003001:Cloning, Molecular; D006224:Cricetinae; D018076:DNA, Complementary / Q000737:chemistry / Q000235:genetics; D006056:Golgi Apparatus / Q000187:drug effects / Q000378:metabolism; D018402:Manduca / Q000737:chemistry / Q000235:genetics*; D008969:Molecular Sequence Data; D052066:N-Ethylmaleimide-Sensitive Proteins; D010802:Phylogeny; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D033921:Vesicular Transport Proteins
D000911:Antibodies, Monoclonal; D002352:Carrier Proteins; D018076:DNA, Complementary; D033921:Vesicular Transport Proteins; C498732:comt protein, Drosophila; D000251:Adenosine Triphosphatases; D052066:N-Ethylmaleimide-Sensitive Proteins