28 Mar 2023
by Richa

Summary- paper 31: Distinct role of TGN-resident clathrin adaptors for Rab5 activation in the TGN-endosome trafficking pathway

Makoto Nagano, Kaito Aoshima, Hiroki Shimamura, Daria Elisabeth Siekhaus, Junko Y. Toshima, Jiro Toshima

Biorxiv, 2023

Questions/gaps addressed:

  • Two types of endosomal compartment in yeast: an early endosome-like compartment containing Rab5 (Vps21p) and a late endosome-like compartment containing Rab7. What is the identity of the Vps21p-residing compartment and how is the compartment generated?

  • GGAs and AP-1 form two distinct clathrin-coated vesicles, the GGA-enriched vesicle, including Ent3 and a minor population of Ent5, and the AP-1-enriched vesicle, which includes most of Ent5 and resides at the late TGN.

  • Activation of yeast Vps21 (Rab5) is partially suppressed by deletion of the epsin-like proteins, Ent3p/5p. Deletion of GGAs or AP-1 complex, or even both, has little effect on Vps21p activity.

  • What are the functional relationships between clathrin adaptors in the Rab5 activation in yeast?

  • Arf1 GTPase and Frq1p, recruit Pik1 to the TGN, which generated PI4P, which recruite GGA first, then AP-1 and Ent5. Ypt31p/32p (Rab11) also regulate the localization of Ent3/5 at the TGN. So Rab11 and Pik1 both regulators?

Key methods:

  • Synthetic genetic interactions test between ent3Δ or ent5Δ mutation with deletion of the GGA1/2 or APL4 (AP-1γ subunit) and looked at Vps21/Rab5 localization. Decrease of GFP-Vps21p fluorescence intensity and an increase in the number of Vps21p compartments when combined ent5Δ with gga1Δ/2Δ. So, GGA-dependent recruitment of Ent3p is important for formation of the Vps21p compartment. AP-1, as well as Ent3p/5p, play a role in formation of the Vps21p compartment.

  • Method to quantitatively assess the Rab5 activity using Nano luciferase fused to either a nanobody that recognizes GFP or to a fragment of human Rabenosyn-5 that binds specifically to the GTP-bound form of Vps21p. Vps21p tagged with GFP-ALFA peptide. Pull from the cell extracts with GST-fused ALFA nanobody, monitoring luciferase activity allows looking at the ratio of active Vps21 to total Vps21. Significantly reduced active Vps21 in ent3Δ ent5Δ apl4Δ vs wt. AP-1 functions redundantly with Ent3p/5p in Vps21p activation.

  • GFP-Vps21 accumulates in clusters near the vacuole in vps9∆, and in ent3Δ ent5Δ apl4Δ. Hse1p (endosomal marker) but not Sec7p (TGN marker) localizes at the Vps21p cluster adjacently to the vacuole. Endosomal characteristics of this cluster? Suppressed by expression of constitutively active Q66L mutant of Vps21p.

  • Role of GGAs, PI(4)P, and yeast Rab11 homologues Ypt31p/32p in the localization of Ent3p/5p and AP-1 at the TGN. GGA1 and GGA2 mutants decreased Ent3 at the TGN, no effect on Ent5 and Apl2. Temperature-sensitive pik1-1(D1055G) mutant, affected all three Ent3, Ent5 and Apl2 (strongest). Rab11 ypt31ts mutant has stronger effect on Ent3, Ent5, weaker on Apl2.

  • Transport of Vps9p from the TGN to the endosome is necessary for Vps21p activation. Shift in punctuate localization of GFP-Vps9p from endosomes to the TGN in the ent3Δ ent5Δ apl4Δ mutant, leading to less active Vps21 at the endosome, and less E-dots. Disruption of Ent3, Ent5 , and Ap-1 progressively suppresses transport of Vps9p from the TGN to the Vps21p compartment. (see model in Fig 7).

Major takeaways:

  • GGA vesicles are formed approximately 10 s prior to the AP-1 vesicles, and GGA vesicles mediate the transport of cargos from the TGN to the vacuole. AP-1 vesicles mediate intra-Golgi recycling.

  • All the three clathrin adaptors, Ent3p/5p, AP-1 and GGAs, contribute to Rab5/Vps21 activation.

  • Ent3p/5p and AP-1 are required for loading Vps9p on the vesicles that transport Vps9p to the Vps21p compartment, while GGAs, together with Pik1p and Rab11s, have a role in the recruitment of Ent3p/5p and AP-1 to the TGN.

  • PI(4)P (Pik1p), Rab11 (Ypt31p/32p), and GGAs are involved in the recruitment of Ent3p to the TGN, function cooperatively recruit Ent3p and contribute to the activation of Vps21p

  • Ent5p is able to complement the function of Ent3p in Rab5 activation. Does Ent5p have dual roles at the TGN: loading of Vps9p onto transport vesicles at the GGA-enriched early TGN, and recycling at the AP-1-enriched late TGN?

  • Recently reported, Tlg2p-residing compartment of the TGN, is distinct from the late TGN where Sec7p resides, is the first destination for endocytic traffic, from which the endocytic cargo is transported to the Vps21p compartment.