First identification and characterization of Shu1 as a cell-surface heme receptor with high-affinity binding (KD ~2.2 μM) essential for heme acquisition in S. pombe

Demonstrates plasma membrane localization via HA4-epitope tagging and fluorescence microscopy, remaining membrane-associated after cell wall digestion

Shows iron-regulated expression with 18-30 fold induction under iron starvation, controlled by Fep1 repressor binding to three GATA elements in the promoter

Structure-function analysis reveals critical cysteine-rich region (Cys72-Cys101) forming partial CFEM-like motif essential for heme binding

Mutant phenotype analysis shows shu1Δ cells cannot grow on heme as sole iron source, demonstrating requirement for heme-iron utilization

Confirms GPI-anchoring at Ser199 through PI-PLC cleavage assays, identifying the ω-site and demonstrating proper membrane attachment

Shows dynamic heme-induced trafficking from plasma membrane (low hemin) to vacuolar membrane (high hemin) using fluorescent heme analog ZnMP

Establishes two-step heme assimilation pathway: Shu1-mediated endocytic uptake into vacuoles followed by Abc3-mediated export to cytosol

Demonstrates that abc3Δ mutants phenocopy shu1Δ, with heme analogs accumulating in vacuoles unable to reach cytoplasm

Demonstrates that the vacuolar ABC transporter Abc3 binds hemin directly through hemin-agarose pulldown assays, establishing Abc3 as the vacuolar hemin exporter that completes the Shu1-dependent heme assimilation pathway (this finding concerns Abc3, not Shu1)

Defines Shu1 as the high-affinity component of a dual heme transport system working alongside low-affinity transporter Str3

Confirms Shu1 requirement for cellular heme import through mutant phenotype analysis

Demonstrates complementary roles of high-affinity (Shu1) and low-affinity (Str3) systems in heme acquisition

Shu1 is an iron-regulated, cell-surface heme-binding protein required for assimilation of exogenous hemin/heme and heme analogs when endogenous heme biosynthesis is compromised (hem1Delta background).

Shu1 binds hemin directly with micromolar affinity (KD ~2.2 uM); spectral titration shows a Soret shift from ~388-391 nm to ~407 nm on binding, and a cysteine quartet (C72/C87/C92/C101) is required for high-affinity binding.

Mutation of the cysteine quartet C72/C87/C92/C101 to alanine strongly reduces hemin-agarose retention and eliminates high-affinity binding, identifying these residues as critical for heme coordination.

Shu1 localizes to the plasma membrane under iron starvation, then is rapidly internalized to the vacuolar membrane after binding hemin/ZnMP, consistent with an endocytic/vacuolar delivery route.

S. pombe has two distinct exogenous heme uptake pathways - Shu1 (a cell-surface GPI-anchored high-affinity route) and Str3 (a distinct MFS transporter route).

In the integrated model, Shu1 is a cell-surface GPI-anchored heme receptor that binds hemin or ZnMP; cargo is delivered to the vacuole and then redistributed to the cytoplasm via the vacuolar ABC transporter Abc3.

Shu1 is specific for heme/porphyrin assimilation and does not substitute for elemental iron uptake; it cannot mediate growth on exogenous FeCl3 alone and heme use is independent of the Fio1/Fip1 oxidase-permease system.

shu1+ transcription is induced by low iron and repressed by iron repletion through the GATA-type repressor Fep1.