Protein Bodies in Leaves Exchange Contents through the Endoplasmic Reticulum

Abstract

Protein bodies (PBs) are organelles found in seeds whose main function is the storage of proteins that are used during germination for sustaining growth. PBs can also be induced to form in leaves when foreign proteins are produced at high levels in the endoplasmic reticulum (ER) and when fused to one of three tags: Zera®, elastin-like polypeptides (ELP), or hydrophobin-I (HFBI). In this study, we investigate the differences between ELP, HFBI and Zera PB formation, packing, and communication. Our results confirm the ER origin of all three fusion-tag-induced PBs. We show that secretory pathway proteins can be sequestered into all types of PBs but with different patterns, and that different fusion tags can target a specific protein to different PBs. Zera PBs are mobile and dependent on actomyosin motility similar to ELP and HFBI PBs. We show in vivo trafficking of proteins between PBs using GFP photoconversion. We also show that protein trafficking between ELP or HFBI PBs is faster and proteins travel further when compared to Zera PBs. Our results indicate that fusion-tag-induced PBs do not represent terminally stored cytosolic organelles, but that they form in, and remain part of the ER, and dynamically communicate with each other via the ER. We hypothesize that the previously documented PB mobility along the actin cytoskeleton is associated with ER movement rather than independent streaming of detached organelles.

LALF32‐51‐E7, a HPV‐16 therapeutic vaccine candidate, forms protein body‐like structures when expressed in Nicotiana benthamiana leaves

“…These findings suggest that the LALF 32-51 peptide is a potential PB-inducer. If this is the case, LALF 32-51 could have significant promise as a fusion partner which enhances the accumulation of other recombinant proteins in plant expression systems, similar to the Zera â peptide, elastin-like polypeptides (ELP) and hydrophobin (Conley et al, 2011;Saberianfar et al, 2016). Its small size is also an advantage.…”

Protein bodies: how the ER deals with high accumulation of recombinant proteins

“…This was confirmed with co‐expression of secretory GFP or ER‐targeted GFP with Zera®‐DsRed in which the GFP signal localized to the periphery of Zera® PBs and not in their core, and explains why Zera®‐DsRed has no significant effect on EPO accumulation (Saberianfar et al ., 2016). …”

“…In the case of Zera®, protein movement between PBs was also observed, but was much slower, likely due to covalent disulphide bridges between Zera® molecules leading to large aggregates (Saberianfar et al ., 2016). These results indicate that PBs are linked to each other through the ER and that PBs are not terminally stored cytosolic organelles.…”

“…The movement of PBs induced by Zera®, ELP and HFBI fusion tags was disrupted by the use of Latrunculin B, an actin depolymerizing drug, and ELP‐induced PBs were shown to lose mobility in the presence of a mutant myosin XI‐K tail. Furthermore, PBs form and align along actin strands and are surrounded by an ER membrane (Conley et al ., 2009; Saberianfar et al ., 2016). Therefore, we speculate that PBs remain connected to the ER, and move along the ER network which relies on the actin cytoskeleton for movement (Figure 1).…”

“…PBs induced by these processes are numerous, round and clustered, they remain in the ER, and they are not destined to the central vacuole (Llop‐Tous et al ., 2010; Saberianfar et al ., 2015, 2016). …”