Masami Matsubae

Mouse Apg16L, a novel WD-repeat protein, targets to the autophagic isolation membrane with the Apg12-Apg5 conjugate

Macroautophagy is the major intracellular degradation system delivering cytoplasmic components to the lysosome/vacuole. We have shown that, in yeast and mammalian cells, the Apg12-Apg5 protein conjugate, which is formed by a ubiquitin-like system, is essential for autophagosome formation. In yeast, the Apg12-Apg5 conjugate interacts with a small coiled-coil protein, Apg16, to form a ∼350 kDa multimeric complex. We demonstrate that the mouse Apg12-Apg5 conjugate forms a ∼800 kDa protein complex containing a novel WD-repeat protein. Because the N-terminal region of this novel protein shows homology with yeast Apg16, we have designated it mouse Apg16-like protein (Apg16L). Apg16L, however, has a large C-terminal domain containing seven WD repeats that is absent from yeast Apg16. Apg16L interacts with both Apg5 and additional Apg16L monomers; neither interaction, however, depends on the WD-repeat domain. In conjunction with Apg12-Apg5, Apg16L associates with the autophagic isolation membrane for the duration of autophagosome formation. Because these features are similar to yeast Apg16, we concluded Apg16L is the functional counterpart of the yeast Apg16. We also found that membrane targeting of Apg16L requires Apg5 but not Apg12. Because WD-repeat proteins provide a platform for protein-protein interactions, the ∼800 kDa complex is expected to function in autophagosome formation, further interacting with other proteins in mammalian cells.

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.

Determination of endogenous peptides in the porcine brain: possible construction of Peptidome, a fact database for endogenous peptides

Peptides play crucial roles in many physiological events. However, a database for endogenous peptides has not yet been developed, because the peptides are easily degraded by proteolytic enzymes during extraction and purification. In this study, we demonstrated that the data for endogenous peptides could be collected by minimizing the proteolytic degradation. We separated porcine brain peptides into 5250 fractions by 2-dimensional chromatography (first ion-exchange and second reversed-phase high-performance liquid chromatography), and 75 fractions of average peptide contents were analyzed in detail by mass spectrometers and a protein sequencer. Based on the analysis data obtained in this study, more than 10000 peptides were deduced to be detected, and more than 1000 peptides to be identified starting from 2 g of brain tissue. Thus, we deduce that it is possible to construct a database for endogenous peptides starting from a gram level of tissue by using 2-dimensional high-performance liquid chromatography coupled with a mass spectrometer.

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 α. These findings suggest that mLANP‐L is transported into the nucleus by Rch1 and/or NPI‐1.

Identification of a novel p300-specific-associating protein, PRS1 (phosphoribosylpyrophosphate synthetase subunit 1)

CBP [CREB (cAMP-response-element-binding protein)-binding protein] and p300 play critical roles in transcriptional co-activation, cell differentiation, proliferation and apoptosis. Multiple transcription factors associate with CBP/p300. With the exception of the SYT oncoprotein, no proteins have been identified that specifically associate with p300, but not CBP. In the present study, we isolated a novel p300-associated protein for which no interaction with CBP was observed by GST (glutathione S-transferase) pull-down assay using Jurkat cell lysates metabolically labelled with [35S]methionine. This protein bound the KIX (kinase-inducible) domain of p300. Following resolution by two-dimensional acrylamide gel electrophoresis, we identified the KIX-domain-bound protein by MS analysis as PRS1 (phosphoribosylpyrophosphate synthetase subunit 1), a protein essential for nucleoside biosynthesis. This is the first report to demonstrate the existence of a p300 KIX-domain-specific-interacting protein that does not interact with CBP. Thus p300 may play a role in the regulation of DNA synthesis through interactions with PRS1.