Probing the plant endomembrane-secretory pathway using heterologous membrane protein markers.

Abstract

Using gene expression systems developed in Nicotiana tabacum, the transport of various heterologous membrane proteins (SYNVG, IBV M, VSVG, and their derivatives) was studied within the plant secretory pathway. The results of these experiments indicated that the proteins accumulated in various endomembrane compartments of plant cells, but not in those counterparts observed in their native hosts. These results suggest that membrane proteins of mammalian and plant cells share a well-conserved mechanism for ER targeting and translocation, but differences exist in their machinery for retention and sorting within the secretory pathway. Part of the project was aimed towards the immuno-isolation of the plant Golgi (or its sub-cisternal elements) using heterologous expression of IBV M and its derivatives. The IBV M molecule was found to be undetectable in transfected protoplasts or transgenic plants. Two chimeric derivatives of IBV M, although stably expressed, were found to be targeted to compartments other than the Golgi. Thus, immuno-isolation of the plant Golgi could not be achieved using this approach. IBV M-GUS (an IBV M derivative) was found to accumulate exclusively in a novel membrane proliferation of the ER, termed an "E-whorl". E-whorls appear devoid of ER-resident enzymes, and their production causes no developmental abnormalities in transgenic plants. A model has been developed in which homo-oligomeric assembly following biosynthesis, is sufficient to prevent forward movement of the IBV M-GUS chimera to the Golgi. Homo-oligomerization also serves to sequester the chimera away from ER resident proteins, and drives the generation of the novel membrane compartment. E-whorls might provide a useful tool for selective accumulation of important proteins that are unstable or insoluble within or toxic to host cells, as well as for amplification of the membrane compartment housing the proteins. Gm 1 (another IBV M derivative) was found to be transported into the plant tonoplast, via a trafficking route from the ER and Golgi to the vacuole. Gm 1 has no lysosomal (or vacuolar) sorting information in either mammalian or plant cells; its targeting to the plant vacuole suggests that an alternative default secretion pathway might exist for the transport of membrane proteins, as reported in yeast.