No abstract

Two cytoplasmic mRNA‐decay pathways have been characterized in yeast, and both are initiated by shortening of the 3′‐poly(A) tail. In the major 5′‐to‐3′ decay pathway, the deadenylation triggers removal of the 5′‐cap, exposing the transcript body for 5′‐to‐3′ degradation. An alternative 3′‐to‐5′ decay pathway also follows the deadenylation and requires two multi‐complexes: the exosome containing various 3′‐exonucleases and the Ski complex consisting of the RNA helicase Ski2p, Ski3p and Ski8p. In addition, Ski7p, which has an N‐terminal domain and a C‐terminal elongation factor 1α‐like GTP‐binding domain, is involved in the 3′‐to‐5′ decay. However, physical interaction between the exosome and the Ski complex, together with the function of Ski7p, has remained unknown. Here we report that the N domain of Ski7p is required and sufficient for the 3′‐to‐5′ decay. Furthermore, the exosome and the Ski complex interact with the different regions of Ski7p N domain, and both interactions are required for the 3′‐to‐5′ decay. Thus, Ski7p G protein appears to function as a signal‐coupling factor between the two multi‐complexes operating in the 3′‐to‐5′ mRNA‐decay pathway.

The small GTPase Rab5, which cycles between active (GTP-bound) and inactive (GDP-bound) states, plays essential roles in membrane budding and trafficking in the early endocytic pathway. However, the molecular mechanisms underlying the Rab5-regulated processes are not fully understood other than the targeting event to early endosomes. Here, we report a novel Rab5-binding protein, RIN3, that contains many functional domains shared with other RIN members and additional Pro-rich domains. RIN3 displays the same biochemical properties as RIN2, the stimulator and stabilizer of GTP-Rab5. In addition, RIN3 exhibits its unique intracellular localization. RIN3 expressed in HeLa cells localized to cytoplasmic vesicles and the RIN3-positive vesicles contained Rab5 but not the early endosomal marker EEA1. Transferrin appeared to be transported partly through the RIN3-positive vesicles to early endosomes. RIN3 was also capable of interacting via its Pro-rich domain with amphiphysin II, which contains SH3 domain and participates in receptor-mediated endocytosis. Interestingly, cytoplasmic amphiphysin II was translocated into the RIN3- and Rab5-positive vesicles when co-expressed with RIN3. These results indicate that RIN3 biochemically characterized as the stimulator and stabilizer for GTP-Rab5 plays an important role in the transport pathway from plasma membrane to early endosomes.

Autophagy is an intracellular degradation pathway conserved in eukaryotes. Among core autophagy-related (Atg) proteins, mammalian Atg9A is the sole multi-spanning transmembrane protein, and both of its N-and C-terminal domains are exposed to the cytoplasm. It is known that Atg9A travels through the trans-Golgi network (TGN) and the endosomal system under nutrient-rich conditions, and transiently localizes to the autophagosome upon autophagy induction. However, the significance of Atg9A trafficking for autophagosome formation remains elusive. Here, we identified sorting motifs in the N-terminal cytosolic stretch of Atg9A that interact with the adaptor protein AP-2. Atg9A with mutations in the sorting motifs could not execute autophagy and was abnormally accumulated at the recycling endosomes. The combination of defects in autophagy and Atg9A accumulation in the recycling endosomes was also found upon the knockdown of TRAPPC8, a specific subunit of the TRAPPIII complex. These results show directly that the trafficking of Atg9A through the recycling endosomes is an essential step for autophagosome formation.

Atg38 provides a physical linkage between the Vps15–Vps34 and Atg14–Vps30 subcomplexes to facilitate PI3-kinase complex I formation.