Redundancy in the insertion pathways

Several studies have suggested the existence of other post-translational pathways operating in the ER-targeting of TA-proteins (Fig. 9, Fig. 10). Some of these pathways overlap in their substrate spectra and may compensate in the targeting of certain TA-proteins. The following pathways are described in the literature:

1.3.4.1. EMC pathway

In a recent study, calmodulin (CaM) was identified to interact with the TA-protein squalene synthase (SQS) (Guna et al. 2018). SQS is a TA-protein that presents a TMD with moderate hydrophobicity. CaM was found to shield TA-proteins with low-hydrophobic TMDs (Guna et al. 2018) (Fig. 10E). Likewise, CaM was reported to interact with TA-proteins in another study (Haßdenteufel et al. 2011). In addition, CaM was also shown to bind hydrophobic regions (Shao and Hegde 2011). CaM was proposed to deliver the TA-protein to the ER membrane protein complex (EMC).

Interestingly, EMC was found to be an ER-insertase for moderately hydrophobic TMDs (Guna et al. 2018) (Fig. 10E). In contrast, CaM was shown to inhibit the ER-insertion of certain TA-proteins in an insertion assay using rabbit reticulocyte lysate (RRL) and rough microsomes (RMs) (Haßdenteufel et al. 2011). Some TA-proteins were shown to have partial dependence on the EMC pathway and in the TRC pathway (Guna et al.

23 2018). Based on those results, the authors proposed an approximate point where both pathways might overlap around the Sec61b TMD hydrophobicity (Guna et al. 2018).

1.3.4.2. PEX pathway

This pathway is responsible of peroxisomal membrane proteins (PMPs) targeting to the ER-membrane or to preexisting peroxisomes (Jones, Morrell, and Gould 2004; reviewed in Mayerhofer 2016). Peroxin-19 (PEX19) is the cytoplasmic factor that recognizes a peroxisomal targeting sequence (PTS) (Gould et al. 1989;

Swinkels et al. 1991) in the PMPs and targets them to the receptor PEX3 (Muntau et al. 2003; Fang et al. 2004; Jones, Morrell, and Gould 2004; Yuqiong Liu, Yagita, and Fujiki 2016) (Fig. 9A, Fig. 10D). PEX3 is a membrane protein localized in the ER and peroxisomes (Toro et al. 2007; Aranovich et al. 2014; Mayerhofer et al. 2016; Schrul and Kopito 2016). The pathway is conserved in yeast and mammals. There are seven PMPs that are TA-proteins (Table 17). The PMPs TA-proteins are targeted to peroxisomes using this pathway. However, Pex15p is targeted using the GET pathway in yeast (van der Zand, Braakman, and Tabak 2010). In contrast, the insertion of the functional homolog of Pex15p in mammals, PEX26, is TRC-independent (Halbach et al. 2006; Yagita, Hiromasa, and Fujiki 2013; Buentzel et al. 2015). The presence of PTS and basic residues following the TMD are responsible for the PEX19-targeting of the PMP TA-proteins (Yagita, Hiromasa, and Fujiki 2013).

1.3.4.3. SND pathway

The SRP-independent targeting (SND) pathway was recently described in yeast (Aviram et al. 2016). SND components described were the cytoplasmic Snd1 and the ER-resident proteins Snd2 and Snd3 (Fig. 9B). Snd1 was predicted to bind the RNC (Fleischer et al. 2006) whereas Snd2 and Snd3 are found in a complex with the translocon (Aviram et al. 2016). The SND pathway predominantly targets membrane proteins whose transmembrane segments are in the middle of the protein. The SND pathway has been shown to compensate for the loss of the SRP pathway and the GET

24

pathway (Aviram et al. 2016), acting as a rescue pathway. In mammals, Snd2 (also known as TMEM208), homolog of the homonym yeast protein, is the only conserved protein from the yeast SND pathway. So far, there are no reported pathway-partners for Snd2 (Fig. 10F) (Yuanbo Zhao et al. 2013; Haßdenteufel et al. 2017).

1.3.4.4. Ubiquilins

Ubiquilins (UBQLN1-4) were reported to be able to chaperone mitochondrial membrane proteins in cytoplasm (Itakura et al. 2016). Mitochondrial TA-proteins are suitable to be UBQLN-substrates (Fig. 10A). In addition, they can also triage these membrane proteins and target them for degradation (Itakura et al. 2016).

1.3.4.5. Hsp40/Hsc70

In a reconstituted system, Hsp40/Hsc70 are able to promote the membrane-insertion of TA-proteins (B. M. Abell et al. 2007; Rabu et al. 2008). However, when tested in HeLa cells in the presence of selective inhibitors of Hsp40/Hsc70 only a small subset of TA-proteins, characterized by low hydrophobicity in their TMDs, was affected (Rabu et al. 2008) (Fig. 9D, Fig. 10B).

1.3.4.6. SRP pathway

VAMP2 and Sec61b in vitro insertion was reported to be SRP-dependent in a post-translational manner (Benjamin M. Abell et al. 2004; B. M. Abell et al. 2007).

Likewise, SRa-downregulated HeLa M cells showed a decrease of SERP1 and Sec61b steady-state levels (Casson et al. 2017). This would suggest that some TA-protein might require the SRP pathway to be targeted to the ER (Casson et al. 2017) (Fig. 10G). Interestingly, Get4-Get5 have been shown to compete for co-translationally-inserted substrates with SRP in a Sgt2-independent way (Zhang et al.

2016).

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Figure 9. Tail-anchored protein insertion pathways in yeast. (A) Pex19 targets PMPs that are TA-proteins with basic residues in its C-terminal tail to its receptor Pex3. Pex3 can be localized in ER or peroxisomes. (B) In the SND pathway, Snd1 can take TA-proteins to its receptor Snd2/Snd3 that forms a complex Sec66, Sec62, Sec72, Sec63 and Sec61. (C) The GET pathway begins when Sgt2 grabs the TA once exits the ribosome, it binds the pre-targeting complex composed of Get4 and Get5 and it hands the TA off to Get3. Get3 is the cytoplasmic factor that carries the protein to the Get1/Get2 ER-receptor and it releases it in an ATP-dependent manner. The receptor is an insertase that inserts the protein into the ER. (D) Hsp70/Hsp40 have been proposed as alternative cytoplasmic factors that can hold TA-proteins. TA-protein model is PLN (PDB ID: 2LPF).

Sgt2

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Figure 10. Tail-anchored protein insertion pathways in mammals. (A) Ubiquilins are believed to target mitochondrial TA-proteins. (B) Hsp70/Hsp40, in a similar fashion to yeast, have hypothetically been proposed as alternative cytoplasmic factors that can hold TA-proteins. (C) The TRC pathway is analog to the Get pathway in yeast but with the addition of the mammalian protein BAG6. SGTA grabs the TA once exits the ribosome, it binds the pre-targeting complex composed of BAG6, UBL4A and TRC35 and it hands the TA off to TRC40. TRC40 is the cytoplasmic factor that carries the protein to the WRB/CAML ER-receptor and it releases it in an ATP-dependent manner. The receptor is an insertase that inserts the protein into the ER. (D) The PEX19 pathway is analog to the one in yeast. PEX19 targets PMPs that are TA-proteins with basic residues in its C-terminal tail to its receptor PEX3 such as PEX26.

PEX3 can be localized in ER or peroxisomes. (E) Calmodulin can target TA-proteins that contain a TMD with low hydrophobicity to the ER membrane protein complex (EMC) that is formed by 10 subunits. (F) Snd2, homolog of the homonym yeast protein, is the only conserved protein in mammals of the yeast SND pathway. There are no reported pathway-partners for Snd2. (G) Some TA-protein might require the SRP pathway to be post-translationally targeted to the ER. TA-protein model is PLN (PDB ID: 2LPF).

BAG6

27 1.3.4.7. Unassisted insertion of TA-proteins

Cytochrome b5 (Cytb5) is a TA-protein that is localized at the ER-membrane. It was the first TA-protein studied (Anderson, Mostov, and Blobel 1983). Cytb5 has a low-hydrophobicity TMD. It has been reported that can be inserted into protein-free liposomes in an unassisted-manner (Yabal et al. 2003; Brambillasca et al. 2005;

Brambillasca et al. 2006; Sara F. Colombo, Longhi, and Borgese 2009). In addition, it might require Hsp40/Hsc70 chaperoning (Rabu et al. 2008). In a similar line, Cytb5 has been reported not to require the TRC pathway for ER-targeting (Stefanovic and Hegde 2007; Favaloro et al. 2008). Cytb5 localized in MOM in cytosol-free semipermeabilized cells (Figueiredo Costa et al. 2018). Therefore, it was proposed that MOM might be the default destination of TA-proteins able to be inserted in an unassisted-manner (Figueiredo Costa et al. 2018).