Tepsin binds LC3B to promote ATG9A trafficking and delivery.

Tepsin is an established accessory protein found in Adaptor Protein 4 (AP-4) coated vesicles, but the biological role of tepsin remains unknown. AP-4 vesicles originate at the trans-Golgi network (TGN) and target the delivery of ATG9A, a scramblase required for autophagosome biogenesis, to the cell periphery. Using in silico methods, we identified a putative LC3-Interacting Region (LIR) motif in tepsin. Biochemical experiments using purified recombinant proteins indicate tepsin directly binds LC3B preferentially over other members of the mammalian ATG8 family. Calorimetry and structural modeling data indicate this interaction occurs with micromolar affinity using the established LC3B LIR docking site. Loss of tepsin in cultured cells dysregulates ATG9A export from the TGN as well as ATG9A distribution at the cell periphery. Tepsin depletion in a mRFP-GFP-LC3B HeLa reporter cell line using siRNA knockdown increases autophagosome volume and number, but does not appear to affect flux through the autophagic pathway. Reintroduction of wild-type tepsin partially rescues ATG9A cargo trafficking defects. In contrast, reintroducing tepsin with a mutated LIR motif or missing N-terminus drives diffuse ATG9A subcellular distribution. Together, these data suggest roles for tepsin in cargo export from the TGN; ensuring delivery of ATG9A-positive vesicles; and in overall maintenance of autophagosome structure.

Statistical Coupling Analysis Predicts Correlated Motions in Dihydrofolate Reductase.

The role of dynamics in enzymatic function is a highly debated topic. Dihydrofolate reductase (DHFR), due to its universality and the depth with which it has been studied, is a model system in this debate. Myriad previous works have identified networks of residues in positions near to and remote from the active site that are involved in dynamics and others that are important for catalysis. For example, specific mutations on the Met20 loop in E. coli DHFR (N23PP/S148A) are known to disrupt millisecond-timescale motions and reduce catalytic activity. However, how and if networks of dynamically coupled residues influence the evolution of DHFR is still an unanswered question. In this study, we first identify, by statistical coupling analysis and molecular dynamic simulations, a network of coevolving residues, which possess increased correlated motions. We then go on to show that allosteric communication in this network is selectively knocked down in N23PP/S148A mutant E. coli DHFR. Finally, we identify two sites in the human DHFR sector which may accommodate the Met20 loop double proline mutation while preserving dynamics. These findings strongly implicate protein dynamics as a driving force for evolution.

Tepsin binds LC3B to promote ATG9A export and delivery at the cell periphery.

Tepsin is an established accessory protein found in Adaptor Protein 4 (AP-4) coated vesicles, but the biological role of tepsin remains unknown. AP-4 vesicles originate at the trans -Golgi network (TGN) and target the delivery of ATG9A, a scramblase required for autophagosome biogenesis, to the cell periphery. Using in silico methods, we identified a putative L C3-Interacting R egion (LIR) motif in tepsin. Biochemical experiments using purified recombinant proteins indicate tepsin directly binds LC3B, but not other members, of the mammalian ATG8 family. Calorimetry and structural modeling data indicate this interaction occurs with micromolar affinity using the established LC3B LIR docking site. Loss of tepsin in cultured cells dysregulates ATG9A export from the TGN as well as ATG9A distribution at the cell periphery. Tepsin depletion in a mRFP-GFP-LC3B HeLa reporter cell line using siRNA knockdown increases autophagosome volume and number, but does not appear to affect flux through the autophagic pathway. Re-introduction of wild-type tepsin partially rescues ATG9A cargo trafficking defects. In contrast, re-introducing tepsin with a mutated LIR motif or missing N-terminus does not fully rescue altered ATG9A subcellular distribution. Together, these data suggest roles for tepsin in cargo export from the TGN; delivery of ATG9A-positive vesicles at the cell periphery; and in overall maintenance of autophagosome structure.

An interaction between ß'-COP and the ArfGAP, Glo3, maintains post-Golgi cargo recycling.

The essential COPI coat mediates retrieval of transmembrane proteins at the Golgi and endosomes following recruitment by the small GTPase, Arf1. ArfGAP proteins regulate COPI coats, but molecular details for COPI recognition by ArfGAPs remain elusive. Biochemical and biophysical data reveal how ß'-COP propeller domains directly engage the yeast ArfGAP, Glo3, with a low micromolar binding affinity. Calorimetry data demonstrate that both ß'-COP propeller domains are required to bind Glo3. An acidic patch on ß'-COP (D437/D450) interacts with Glo3 lysine residues located within the BoCCS (binding of coatomer, cargo, and SNAREs) region. Targeted point mutations in either Glo3 BoCCS or ß'-COP abrogate the interaction in vitro, and loss of the ß'-COP/Glo3 interaction drives Ste2 missorting to the vacuole and aberrant Golgi morphology in budding yeast. These data suggest that cells require the ß'-COP/Glo3 interaction for cargo recycling via endosomes and the TGN, where ß'-COP serves as a molecular platform to coordinate binding to multiple proteins, including Glo3, Arf1, and the COPI F-subcomplex.