Characterization of Inverted Membrane Vesicles from the Halophilic Archaeon Haloferax volcanii

Abstract

Abstract. Membrane-related processes in archaea, the third and most-recently described domain of life, are in general only poorly understood. One obstacle to a functional understanding of archaeal membrane-associated activities corresponds to a lack of archaeal model membrane systems. In the following, characterization of inverted archaeal membrane vesicles, prepared from the halophilic archaeon Haloferax volcanii, is presented. The inverted topology of the vesicles was revealed by defining the orientation of membrane-bound enzymes that in intact cells normally face the cytoplasm or of other protein markers, known to face the exterior medium in intact cells. Electron microscopy, protease protection assays and lectin-binding experiments confirmed the sealed nature of the vesicles. Upon alkalinization of the external medium, the vesicles were able to generate ATP, reflecting the functional nature of the membrane preparation. The availability of preparative scale amounts of inverted archaeal membrane vesicles provides a platform for the study of various membranerelated phenomena in archaea.

Membrane Binding of Ribosomes Occurs at SecYE-based Sites in the Archaea Haloferax volcanii

“…44 In IMVs, however, the enzymatic activity is readily detectable from the medium, and as such can serve as a reporter of the topology of H. volcanii membrane vesicles. 39 In IMVs prepared from wild-type H. volcanii cells, 89% of the normally inward-facing enzyme activity was accessible (Table 1), in agreement with earlier studies. 39 In the case of CBD-SecE-and CBD-SecY-containing IMV preparations, 79% and 90% of the menadionedependent NADH dehydrogenase activity now faced the exterior, respectively.…”

“…39 In IMVs prepared from wild-type H. volcanii cells, 89% of the normally inward-facing enzyme activity was accessible (Table 1), in agreement with earlier studies. 39 In the case of CBD-SecE-and CBD-SecY-containing IMV preparations, 79% and 90% of the menadionedependent NADH dehydrogenase activity now faced the exterior, respectively. However, since the normally sequestered enzyme would be accessible to added substrate in membrane fragments as well as IMVs, the sealed nature of the vesicles was tested.…”

“…41 In the inverted membranes, the S-layer glycoprotein is oriented towards the vesicle lumen and thus protected from externally-added protease. 39 Indeed, proteolytic treatment had no effect on the level or distribution of the internally oriented S-layer glycoprotein present in fractions collected following flotation ( Figure 5C). Hence, the treatment with proteinase K that led to a loss in H. volcanii ribosome binding did not disrupt IMV integrity.…”

“…With both active H. volcanii ribosomes and biologically functional H. volcanii IMVs available, 39 the ability of the ribosomes to interact with the plasma membrane was addressed next. In preliminary studies, ribosomal markers were identified that, in subsequent experiments, could serve to distinguish the ribosomes from the IMVs when the two would be combined.…”

Post-translational Secretion of Fusion Proteins in the Halophilic Archaea Haloferax volcanii

“…Given the availability of inverted membrane vesicles from H. volcanii (43), reconstituted H. volcanii SRP (44), and purified SP-CBD 2 and SP-bacterioopsin-DHFR-1 (45) preproteins, the stage is now set to reconstitute archaeal protein translocation in vitro and to address these questions.…”