[Proteomic analysis using mass spectrometry].
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Amino Acid Sequence↗
Biomedical subjects
M Matsubae
Publications and source records attributed to M Matsubae.
Characterization of the nuclear transport of a novel leucine-rich acidic nuclear protein-like protein.
We previously reported that the nuclear localization signal (NLS) peptides stimulate the in vitro phosphorylation of several proteins, including a 34 kDa protein. In this study, we show that this specific 34 kDa protein is a novel murine leucine-rich acidic nuclear protein (LANP)-like large protein (mLANP-L). mLANP-L was found to have a basic type NLS. The co-injection of Q69LRan-GTP or SV40 T-antigen NLS peptides prevented the nuclear import of mLANP-L. mLANP-L NLS bound preferentially to Rch1 and NPI-1, but not to the Qip1 subfamily of importin alpha. These findings suggest that mLANP-L is transported into the nucleus by Rch1 and/or NPI-1.
Amino Acid Sequence↗
In vivo evidence for involvement of a 58 kDa component of nuclear pore-targeting complex in nuclear protein import.
We recently showed that a nuclear location signal (NLS)-containing karyophile forms a stable complex with cytoplasmic components for nuclear pore-targeting The complex, termed nuclear pore-targeting complex (PTAC), contained two essential proteins of 54 and 90 kDa, respectively, as estimated by electrophoresis. In this study, we found that the 54 kDa component of PTAC is the mouse homologue of Xenopus importin (m-importin). Cytoplasmic injection of the antibodies raised against recombinant m-importin showed an inhibitory effect on nuclear import of a karyophile in living mammalian cells. A portion of cytoplasmically injected antibodies migrated rapidly into the nucleus, indicating dynamic movement of this protein across the nuclear envelope. Moreover, the injected antibodies co-precipitated the karyophile, in an NLS-dependent manner, with endogenous m-importin in the cytoplasm. These results provide in vivo evidence that m-importin is involved in nuclear protein import through association with a NLS in the cytoplasm before nuclear pore binding.
Amino Acid Sequence↗
The nuclear pore-targeting complex binds to nuclear pores after association with a karyophile.
We recently showed that a karyophilic protein forms a stable complex, termed nuclear pore-targeting complex (PTAC), with cytoplasmic components prior to nuclear pore-binding. In this study, we cloned a cDNA encoding a 97 kDa of PTAC (PTAC97). Recombinant PTAC97 completely reconstitutes the nuclear binding-step in conjunction with a 58 kDa component of PTAC (PTAC58) in the semi-intact cell-free transport assay. Biochemical analysis reveals that PTAC58 binds to a karyophilic protein, and PTAC97 is associated with PTAC58 in a 1:1 molar ratio. A complex of PTAC97 and PTAC58 targets nuclear pores, depending on the presence of a karyophile. These in vitro results suggest that the first step in nuclear import occurs through the targeting-complex formation of a karyophile with PTAC58 bound to PTAC97.
Amino Acid Sequence↗
A karyophilic protein forms a stable complex with cytoplasmic components prior to nuclear pore binding.
Targeting of karyophilic proteins to nuclear pores is known to require several cytoplasmic factors, including the nuclear location signal-binding protein. Using a digitonin-permeabilized cell-free transport assay, we have obtained a cytoplasmic fraction containing factors that specifically bind to karyophilic protein and support the nuclear binding step of the transport. Components in this fraction form a stable complex with the karyophile through interaction with nuclear location signal. Since this complex shows nuclear pore binding activity prior to nuclear entry in the absence of other cytosolic factors, we call it nuclear pore-targeting complex. It consists of karyophilic protein and four proteins of 54, 56, 66, and 90 kDa. In our reconstitution experiments, a complex with 54 and 90 kDa proteins is capable of targeting karyophiles to the nuclear pores.
Amino Acid Sequence↗