TY - JOUR

T1 - Human hAtg2A protein expressed in yeast is recruited to preautophagosomal structure but does not complement autophagy defects of atg2Δ strain

AU - Romanyuk, Daria

AU - Polak, Anna

AU - Maleszewska, Agnieszka

AU - Sieńko, Marzena

AU - Grynberg, Marcin

AU - Żołądek, Teresa

PY - 2011

Y1 - 2011

N2 - 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 atg2D 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.

AB - 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 atg2D 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.

KW - Amino Acid Sequence

KW - Aminopeptidases/genetics

KW - Autophagy

KW - Autophagy-Related Proteins

KW - Cytoplasm/metabolism

KW - Genetic Complementation Test

KW - Green Fluorescent Proteins/genetics

KW - Humans

KW - Membrane Proteins/genetics

KW - Molecular Sequence Data

KW - Phagosomes/metabolism

KW - Protein Structure, Tertiary

KW - Protein Transport

KW - Recombinant Fusion Proteins/genetics

KW - Saccharomyces cerevisiae/genetics

KW - Saccharomyces cerevisiae Proteins/genetics

KW - Sequence Homology, Amino Acid

KW - Two-Hybrid System Techniques

KW - Vacuoles/metabolism

M3 - Article

C2 - 21887408

VL - 58

SP - 365

EP - 374

JO - Acta Biochimica Polonica

JF - Acta Biochimica Polonica

SN - 0001-527X

IS - 3

ER -