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The transport of secretory proteins from the endoplasmic reticulum to the vacuole requires sorting signals as well as specific transport mechanisms. This work is focused on the transport in transgenic tobacco (Nicotiana tabacum) plants of a human α-mannosidase, MAN2B1, which is a lysosomal enzyme involved in the turnover of N-linked glycoproteins and can be used in enzyme replacement therapy. Although ubiquitously expressed, α-mannosidases are targeted to lysosomes or vacuoles through different mechanisms according to the organisms in which these proteins are produced. In tobacco cells, MAN2B1 reaches the vacuole even in the absence of mannose-6-phosphate receptors, which are responsible for its transport in animal cells. We report that MAN2B1 is targeted to the vacuole without passing through the Golgi complex. In addition, a vacuolar targeting signal that is recognized in plant cells is located in the MAN2B1 amino-terminal region. Indeed, when this amino-terminal domain is removed, the protein is retained in the endoplasmic reticulum. Moreover, when this domain is added to a plant-secreted protein, the resulting fusion protein is partially redirected to the vacuole. These results strongly suggest the existence in plants of a new type of vacuolar traffic that can be used by leaf cells to transport vacuolar proteins.

The major seed storage proteins of maize (Zea mays) and bean (Phaseolus vulgaris), zein and phaseolin, accumulate in the endoplasmic reticulum (ER) and in storage vacuoles, respectively. We show here that a chimeric protein composed of phaseolin and 89 amino acids of gamma-zein, including the repeated and the Pro-rich domains, maintains the main characteristics of wild-type gamma-zein: It is insoluble unless its disulfide bonds are reduced and forms ER-located protein bodies. Unlike wild-type phaseolin, the protein, which we called zeolin, accumulates to very high amounts in leaves of transgenic tobacco (Nicotiana tabacum). A relevant proportion of the ER chaperone BiP is associated with zeolin protein bodies in an ATP-sensitive fashion. Pulse-chase labeling confirms the high affinity of BiP to insoluble zeolin but indicates that, unlike structurally defective proteins that also extensively interact with BiP, zeolin is highly stable. We conclude that the gamma-zein portion is sufficient to induce the formation of protein bodies also when fused to another protein. Because the storage proteins of cereals and legumes nutritionally complement each other, zeolin can be used as a starting point to produce nutritionally balanced and highly stable chimeric storage proteins.