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Mutational analysis of the AtNUDT7 Nudix hydrolase from Arabidopsis thaliana reveals residues required for protein quarternary structure formation and activity

100%

Olejnik K., Plochocka D., Grynberg M., Goch G., Gruszecki W. I., Basinska T., Kraszewska E.

Acta Biochimica Polonica

2009

tom 56

nr 2

291-300

Arabidopsis thaliana AtNUDT7, a homodimeric Nudix hydrolase active on ADP-ribose and NADH, exerts negative control on the major signaling complex involved in plant defense activation and programmed cell death. The structural and functional consequences of altering several amino-acid residues of the AtNUDT7 protein have been examined by site-directed mutagenesis, far-UV circular dichroism (CD), attenuated total reflection-Fourier transform infrared (ATR-FTIR) and photon correlation (PCS) spectroscopy, biochemical analysis and protein-protein interaction studies. Alanine substitutions of F73 and V168 disallowed dimer formation. Both the F73A- and V168A-mutated proteins displayed no observable enzymatic activity. Alanine substitution of the V69 residue did not significantly alter the enzyme activity and had no influence on dimer arrangement. The non-conserved V26 residue, used as a negative control, did not contribute to the enzyme quaternary structure or activity. Detailed biophysical characterization of the wild-type and mutant proteins indicates that the mutations do not considerably alter the secondary structure of the enzyme but they affect dimer assembly. In addition, mutating residues V69, F73 and V168 disrupted the binding of AtNUDT7 to the regulatory 14.3.3 protein. These are the first studies of the structure-function relationship of AtNUDT7, a Nudix hydrolase of important regulatory function.

Human hAtg2A protein expressed in yeast is recruited to preautophagosomal structure but does not complement autophagy defects of atg2A strain

100%

Romanyuk D., Polak A., Maleszewska A., Sienko M., Grynberg M., Zoladek T.

Acta Biochimica Polonica

2011

tom 58

nr 3

Yeast Atg2, an autophagy-related protein, is highly conserved in other fungi and has two homologues in humans, one of which is hAtg2A encoded by the hATG2A/KIAA0404 gene. Region of homology between Atg2 and hAtg2A proteins comprises the C-terminal domain. We used yeast atg2Δ strain to express the GFP-KIAA0404 gene, its fragment or fusions with yeast ATG2, and study their effects on autophagy. The GFP-hAtg2A protein localized to punctate structures, some of which colocalized with Ape1-RFP-marked preautophagosomal structure (PAS), but it did not restore autophagy in atg2Δ cells. N-terminal fragment of Atg2 and N-terminal fragment of hAtg2A were sufficient for PAS recruitment but were not sufficient to function in autophagy. Neither a fusion of the N-terminal fragment of hAtg2A with C-terminal domain of Atg2 nor a reciprocal fusion were functional in autophagy. hAtg2A, in contrast to yeast Atg2, did not show interaction with the yeast autophagy protein Atg9 but both Atg2 proteins showed interaction with Atg18, a phospholipid-binding protein, in two-hybrid system. Moreover, deletion of ATG18 abrogated PAS recruitment of hAtg2A. Our results show that human hAtg2A can not function in autophagy in yeast, however, it is recruited to the PAS, possibly due to the interaction with Atg18.

Ograniczanie wyników

2 Acta Biochimica Polonica

2 Grynberg M.

1 Basinska T.

1 Goch G.

1 Gruszecki W. I.

1 Kraszewska E.

1 Maleszewska A.

1 Olejnik K.

1 Plochocka D.

1 Polak A.

1 Romanyuk D.

1 Sienko M.

1 Zoladek T.

1 2011

1 2009

Wyniki wyszukiwania

Mutational analysis of the AtNUDT7 Nudix hydrolase from Arabidopsis thaliana reveals residues required for protein quarternary structure formation and activity

Human hAtg2A protein expressed in yeast is recruited to preautophagosomal structure but does not complement autophagy defects of atg2A strain