Spf1 and Ste24: quality controllers of transmembrane protein topology in the eukaryotic cell

Abstract

DNA replication, transcription, and translation in eukaryotic cells occur with decreasing but still high fidelity. In contrast, for the estimated 33% of the human proteome that is inserted as transmembrane (TM) proteins, insertion with a non-functional inverted topology is frequent. Correct topology is essential for function and trafficking to appropriate cellular compartments and is controlled principally by responses to charged residues within 15 residues of the inserted TM domain (TMD); the flank with the higher positive charge remains in the cytosol (inside), following the positive inside rule (PIR). Yeast (Saccharomyces cerevisiae) mutants that increase insertion contrary to the PIR were selected. Mutants with strong phenotypes were found only in SPF1 and STE24 (human cell orthologs are ATP13A1 and ZMPSte24) with, at the time, no known relevant functions. Spf1/Atp13A1 is now known to dislocate to the cytosol TM proteins inserted contrary to the PIR, allowing energy-conserving reinsertion. We hypothesize that Spf1 and Ste24 both recognize the short, positively charged ER luminal peptides of TM proteins inserted contrary to the PIR, accepting these peptides into their large membrane-spanning, water-filled cavities through interaction with their many interior surface negative charges. While entry was demonstrated for Spf1, no published evidence directly demonstrates substrate entry to the Ste24 cavity, internal access to its zinc metalloprotease (ZMP) site, or active withdrawal of fragments, which may be essential for function. Spf1 and Ste24 comprise a PIR quality control system that is conserved in all eukaryotes and presumably evolved in prokaryotic progenitors as they gained differentiated membrane functions. About 75% of the PIR is imposed by this quality control system, which joins the UPR, ERAD, and autophagy (ER-phagy) in coordinated, overlapping quality control of ER protein function.

Keywords: positive inside rule; quality control; topology; topology error recognition; transmembrane proteins.

FIGURE 1

PIR quality control system. (A) If the more positively charged TMD-flanking peptide of a TM protein is the C-terminus, as in SPßla and SPInv fusions, the PIR requires that this flank remains in the cytoplasm (Ccyt/Nexo), in the functional orientation during insertion. (B) Any of this TM protein inserted with inverted topology (Ncyt/Cexo) is non-functional. Approximately half of this inverted protein is accepted by the Spf1 cavity for ATPase-driven dislocation, allowing reinsertion (McKenna et al., 2020; McKenna et al., 2022; center). Most of the rest is eliminated by Ste24. A hypothetical mechanism is the charge-directed entry into the Ste24 cavity and ZMP cleavage, possibly aided by removal by the Dfm1-recruited cdc48 AAA ATPase, followed by proteasomal disassembly (right).

FIGURE 2

Model TM proteins (Tipper and Harley, 2002). The N-terminus of Ste2 (left) is shown with only TMDs 1 and 2. Two N-terminal negative charges and three C-terminal positive charges are all within the eight residues of TMD1, resulting in a charge difference of +5. The SPβla and SPInv constructs are shown in Nexo (center) and Cexo (right) topology. S is the N-terminal 79 residues of Ste2, including its Nexo TMD1 and the first cytoplasmic loop with a single exofacial N-glycosylation site; P is a peptide with two glycosylation sites that are cleaved by Kex2 in the Golgi if the fusion is Cexo. The gel mobilities of the Nexo and Cexo forms of SPβla are 52 and 55 kDa, respectively; their ratio measures the SPβla topology (% Cexo). The SPInv fusion is used for PIR mutant selection; when Cexo is inserted, Golgi cleavage and secretion are efficient, allowing invertase secretion and growth on sucrose plates. An assay of secreted invertase activity, compared to a control construct with almost complete Cexo insertion provides an independent assay of topology.

FIGURE 3

Cryo-EM structures of Spf1. (A) Ribbon representation of apo Spf1 in a conformation open to the cytosol (inward). (B) Surface representation with the “V”-shaped substrate-binding pocket outlined (dashed line); bound substrates can exit the cytosol. (C) Enlarged view of the substrate-binding pocket (light gold surface) with a closed luminal gate. (D) Ribbon representation of BeF3-bound Spf1 in a conformation open to the lumen (outward). (E) Surface representation with the substrate-binding pocket outlined (dashed line). (F)Enlarged view of the substrate-binding pocket (light gold surface) with an open luminal gate. This cavity should preferentially bind ER-TM proteins with positively charged luminal peptides. Reproduced with permission from McKenna et al. (2020).

FIGURE 4

Structure of Ste24p. Ribbon representation of the ß barrel structure of Ste24p (light gray) with a large central cavity of more than 12,000 Å³ represented as a light gold surface. Ste24p is a membrane-bound zinc metalloprotease (ZMP) with seven TM α-helices. Helices VI and VII contain the zinc-binding site (Zn shown as a red sphere). Membrane–cytosol interface domains are shown in magenta (L5D, the loop 5 domain) and in blue (the C-terminal section of the ZMP domain). Reproduced with permission from Goblirsch and Wiener (2020).