Molecular farming aims at producing high-value proteins in plants for pharmaceutical or other industrial use. Tobacco is one of the most used systems in this field of research, offering developed technology for gene transfer and protein expression. Foreign protein stability is a major issue in molecular farming and is strongly dependent on the subcellular compartment of accumulation. We are screening various compartments of the plant endomembrane system for the high accumulation of the HIV-1 p24 nucleocapsid protein, a potential vaccine against the human immunodeficiency virus. Previous work has shown that the localisation of fusions of GFP (Green Fluorescent Protein) to the full length or truncated versions of the transmembrane domain (TMD) of human LAMP1 (Luminal Associated Membrane Protein 1) in tobacco, was detected in the lumen of different compartments of the plant secretory pathway (endoplasmic reticulum -ER-, Golgi Apparatus or plasma membrane), depending on the length of the TMD. In this work, different p24 fusion proteins were designed to accumulate in different compartments of the secretory pathway in tobacco cells. Therefore, the HIV-1 p24 was fused at the N- or C-terminus of the Red Fluorescent Protein (RFP) followed by the different TMDs. Moreover, p24 was also N- or C-terminally fused to the N-terminal domain of maize prolamin γ-zein (zein-p24 and p24-zein). Zein proteins are originally accumulated in ER-derived protein bodies in seeds and previous studies showed the potential of accumulating heterologous proteins in this compartment protecting them from proteases and enhancing their stability. Finally, p24 was fused to the C-terminal tail-anchor of cytochrome b5 (p24-TA), which is expected to be anchored in the ER membrane facing the cytosol. Localisation studies in tobacco protoplasts showed that the constructs containing RFP at the C-terminus of the p24 (p24RFP-TMD) are targeted to the expected compartments (ER, Golgi Apparatus or plasma membrane). However, when the RFP is placed at the N-terminus of p24 (RFPp24-TMD) the fluorescence appears in the tonoplast and the vacuolar lumen, indicating vacuolar delivery and cleavage from the membrane anchor. Transgenic tobacco plants expressing the p24RFP-TMD fusion proteins with the correct targeting to the ER, Golgi and plasma membrane, and also expressing zein-p24, p24-zein and p24-TA were produced. The highest accumulation levels (1% of total soluble protein, TSP) were achieved for p24 containing zein in either N-terminal or Cterminal position. Fusion proteins targeted to the ER showed different accumulation levels if the protein was exposed on the luminal side (p24RFP-TMD, 0.3% TSP) or in the cytosolic side (p24-TA, 0.15% TSP). p24RFP-TMD fusion proteins accumulating in the Golgi apparatus and the plasma membrane showed accumulation levels around 0.15% TSP. The zein fusions formed polymers that were in part difficult to denature even in the presence of SDS, a feature that suggests protein body formation. Pulse-chase experiments indicated that the difference in accumulation of the constructs was mainly due to difference in protein stability. However, RNA blot analysis showed that the zein fusions also lead to increased RNA accumulation. In all cases, the p24 could be released from the fusion tags by digestion with thrombin as the p24 fusion proteins were designed to have a thrombin cleavage site for purification purposes. On the whole, these results highlight the promising approach of targeting HIV-1 p24 to the ER by fusing to the zein domain and provide new information on the relationship between subcellular localisation and stability of integral membrane proteins
