Light-driven proton-pumping rhodopsins are widely distributed in many microorganisms. They convert sunlight energy into proton gradients that serve as energy source of the cell. Here we report a new functional class of a microbial rhodopsin, a light-driven sodium ion pump. We discover that the marine flavobacterium Krokinobacter eikastus possesses two rhodopsins, the first, KR1, being a prototypical proton pump, while the second, KR2, pumps sodium ions outward. Rhodopsin KR2 can also pump lithium ions, but converts to a proton pump when presented with potassium chloride or salts of larger cations. These data indicate that KR2 is a compatible sodium ion-proton pump, and spectroscopic analysis showed it binds sodium ions in its extracellular domain. These findings suggest that light-driven sodium pumps may be as important in situ as their proton-pumping counterparts.

Yeast extract (0.025%) and nalidixic acid (0.002%) were added to seawater samples and the samples were incubated for 6 h at 20 degrees C in the dark. Under these conditions, bacterial cells did not divide but grew to form elongated cells that are easily recognized by a direct microscopic method and epifluorescent microscopic technique. The number of cells thus obtained is proposed as a direct cound of viable bacterial cells (DVC). With open ocean samples, DVC was higher than 'viable' plate counts by up to three orders of magnitude and lower than the direct counts by about one order.

Krokinobacter eikastus rhodopsin 2 (KR2) is the first light-driven Na(+) pump discovered, and is viewed as a potential next-generation optogenetics tool. Since the positively charged Schiff base proton, located within the ion-conducting pathway of all light-driven ion pumps, was thought to prohibit the transport of a non-proton cation, the discovery of KR2 raised the question of how it achieves Na(+) transport. Here we present crystal structures of KR2 under neutral and acidic conditions, which represent the resting and M-like intermediate states, respectively. Structural and spectroscopic analyses revealed the gating mechanism, whereby the flipping of Asp116 sequesters the Schiff base proton from the conducting pathway to facilitate Na(+) transport. Together with the structure-based engineering of the first light-driven K(+) pumps, electrophysiological assays in mammalian neurons and behavioural assays in a nematode, our studies reveal the molecular basis for light-driven non-proton cation pumps and thus provide a framework that may advance the development of next-generation optogenetics.

LETTERS TO NATURE membrane proteins) between the cisternae is thought to occur mainly at the dilated rims of the Oolgi stacks 22 • 23 • If Rab6p were involved in this process, it would be expected to be more concentrated at the edges of the cisternae. But the exact topology of intra-Oolgi traffic has been very difficult to examine in vivo and there is no strong evidence to support this model. Alternatively, Rab6p could be involved in an as-yet-unknown transport event between the Oolgi stacks.Finally, the observation of the polarized distribution of Rab6p in the Oolgi apparatus is of interest. This suggests that another OTP-binding protein may act in intra-Oolgi transport before Rab6p. Such a protein could be Rablp, the mammalian counterpart of yeast Yptlp (refs 5,12,13). Our results support the hypothesis that several small OTP-binding proteins localize to different intracellular compartments and have a pivotal role in maintaining the orderly flow of vesicular traffic in mammalian ~~-D

Light-activated, ion-pumping rhodopsins are broadly distributed among many different bacteria and archaea inhabiting the photic zone of aquatic environments. Bacterial proton-or sodium-translocating rhodopsins can convert light energy into a chemiosmotic force that can be converted into cellular biochemical energy, and thus represent a widespread alternative form of photoheterotrophy. Here we report that the genome of the marine flavobacterium Nonlabens marinus S1-08 T encodes three different types of rhodopsins: Nonlabens marinus rhodopsin 1 (NM-R1), Nonlabens marinus rhodopsin 2 (NM-R2), and Nonlabens marinus rhodopsin 3 (NM-R3). Our functional analysis demonstrated that NM-R1 and NM-R2 are light-driven outward-translocating H + and Na + pumps, respectively. Functional analyses further revealed that the lightactivated NM-R3 rhodopsin pumps Cl − ions into the cell, representing the first chloride-pumping rhodopsin uncovered in a marine bacterium. Phylogenetic analysis revealed that NM-R3 belongs to a distinct phylogenetic lineage quite distant from archaeal inward Cl − -pumping rhodopsins like halorhodopsin, suggesting that different types of chloride-pumping rhodopsins have evolved independently within marine bacterial lineages. Taken together, our data suggest that similar to haloarchaea, a considerable variety of rhodopsin types with different ion specificities have evolved in marine bacteria, with individual marine strains containing as many as three functionally different rhodopsins.