Evidence That Subcellular Localization of a Bacterial Membrane Protein Is Achieved by Diffusion and Capture

Evidence that subcellular localization of a bacterial membrane protein is achieved by diffusion and capture David Z. Rudner, Qi Pan, and Richard M. Losick* Department of Molecular and Cellular Biology, Harvard University, 16 Divinity Avenue, Cambridge, MA 02138 Contributed by Richard M. Losick, April 16, 2002 Bacteria lack an endoplasmic reticulum, a Golgi apparatus, and uted uniformly throughout the cell. In the third model, which we transport vesicles and yet are capable of sorting and delivering call diffusion and capture, the protein is inserted randomly into integral membrane proteins to particular sites within the cell with all membranes and becomes localized by diffusion to, and high precision. What is the pathway by which membrane proteins capture at, the site where it will ultimately reside. reach their proper subcellular destination in bacteria? We have An attractive system in which to address the question of how addressed this question by using green fluorescent protein (GFP) membrane proteins localize is the process of sporulation in B. fused to a polytopic membrane protein (SpoIVFB) that is involved subtilis. Sporulation takes place in two principal morphological in the process of sporulation in the bacterium Bacillus subtilis. stages (7). In the first stage, the sporulating cell (or sporangium) SpoIVFB-GFP localizes to a region of the sporulating cell known as divides asymmetrically through the formation of a polar septum, the outer forespore membrane, which is distinct from the cyto- which creates a small cell known as the forespore and a larger cell plasmic membrane. Experiments are presented that rule out a called the mother cell. Initially, the forespore and mother cell lie mechanism in which SpoIVFB-GFP localizes to all membranes but is side-by-side, but in the second stage of development the fore- selectively eliminated from the cytoplasmic membrane by proteo- spore is engulfed by the mother cell. Engulfment is a phagocytic- lytic degradation and argue against a model in which SpoIVFB-GFP like process in which the membranes of the polar septum migrate is selectively inserted into the outer forespore membrane. Instead, around the forespore, pinching it off as a free protoplast within the results are most easily compatible with a model in which the mother cell. Engulfment eventually creates two topologically SpoIVFB-GFP achieves proper localization by insertion into the distinct membranes within the mother cell: the cytoplasmic cytoplasmic membrane followed by diffusion to, and capture in, the outer forespore membrane. The possibility that diffusion and membrane, which delimits the periphery of the cell, and the capture is a general feature of protein localization in bacteria is mother cell membrane that surrounds the forespore (also known discussed. as the outer forespore membrane). Several proteins have been identified that localize specifically to the outer forespore mem- sporulation brane, one of the best-studied examples is the sporulation protein SpoIVFB (8–10). SpoIVFB is a proprotein processing enzyme that is involved espite their small size and apparent simplicity, bacteria in the activation of a transcription factor that acts at a late stage Dexhibit a high degree of subcellular organization in which of development (11–14). SpoIVFB, which has six transmem- proteins frequently localize to particular sites within the cell (1). brane segments (15), is synthesized in the mother cell during the Examples include the cell division protein FtsZ, which assembles process of engulfment (8). That SpoIVFB exhibits a highly into a cytokinetic ring at the mid cell of Escherichia coli (2); the specific pattern of subcellular localization is seen through the use chemotaxis receptor McpA, which localizes to the cell pole in Caulobacter crescentus (3); the membrane phosphatase SpoIIE, of a functional fusion of the sporulation protein to the green which localizes to the sporulation septum in Bacillus subtilis (4); fluorescent protein (GFP) (ref. 9; Fig. 1A). Fluorescence from and the actin polymerization protein IcsA, which localizes to the SpoIVFB-GFP is coincident with the outer forespore membrane older of the two cell poles of Shigella flexneri (5). Here, we are with little or no signal detectable at the cytoplasmic membrane concerned with the problem of how integral membrane proteins (9, 10) (Fig. 1A). Proper localization of SpoIVFB-GFP depends become localized within the bacterial cell. Unlike eukaryotes, on a second integral membrane protein (SpoIVFA) that resides bacteria lack organelles and membrane vesicles that sort and in a complex with SpoIVFB-GFP (10). In the absence of SpoIVFA, SpoIVFB-GFP localizes uniformly to all membranes deliver membrane proteins to particular subcellular addresses. CELL BIOLOGY Thus, an outstanding question in the field of prokaryotic biology within the mother cell (10). SpoIVFA also localizes to the outer is the nature of the pathway(s) by which integral membrane forespore membrane and does so in a manner that does not proteins reach their proper destination inside the cell. depend on SpoIVFB (10). We envision three models for how bacterial membrane pro- SpoIVFB-GFP could achieve proper localization to the outer teins become properly localized. In the first model, which we call forespore membrane by any of the three models we have targeted insertion, the protein is directly and selectively inserted presented (Fig. 1B). It could be inserted directly and selectively into the membrane at the site where it will ultimately reside. This into the mother-cell membrane that surrounds the forespore. model has been proposed for the mechanism by which the outer This targeted insertion could occur cotranslationally or post- membrane protein IcsA achieves proper localization at the old translationally as shown for simplicity in Fig. 1B. Alternatively, cell pole in Shigella (5, 6). The second model, which we call SpoIVFB-GFP could be inserted randomly into all mother-cell selective degradation, involves the random insertion of the membranes and then selectively degraded from the cytoplasmic protein into all membranes in the cell followed by proteolytic membrane. Finally, SpoIVFB-GFP could be inserted into all elimination of the protein from sites other than its proper membranes and diffuse to the engulfing mother-cell membrane destination. Selective degradation could be achieved by a protease with a spatially restricted distribution in the cell or by an alternative mechanism in which the protein is protected from Abbreviation: GFP, green fluorescent protein. degradation at its proper location by a protease that is distrib- *To whom reprint requests should be addressed. E-mail: [email protected]. www.pnas.org͞cgi͞doi͞10.1073͞pnas.132235899 PNAS ͉ June 25, 2002 ͉ vol. 99 ͉ no. 13 ͉ 8701–8706 Downloaded by guest on October 1, 2021 Fig. 1. The sporulation membrane protein SpoIVFB-GFP localizes to the mother-cell membrane that engulfs the forespore. (A) Model of the polytopic membrane protein SpoIVFB fused to GFP (green ball). Fluorescent micrograph of SpoIVFB-GFP from strain BDR497 at hour 2.5 of sporulation. SpoIVFB-GFPis synthesized in the mother-cell compartment and localizes to the mother-cell membrane that surrounds the forespore. Membranes (false-colored red) were labeled with TMA-DPH at the start of sporulation. The asymmetric septa and membranes engulfing the forespore stain more intensely because they are composed of two layers of membrane. (Bar ϭ 1 ␮m.) (B) Three models for how SpoIVFB-GFP achieves proper localization to the engulfing septal membrane. In the first model, SpoIVFB-GFP is inserted directly into the septal membrane. In the second and third models, SpoIVFB-GFP is randomly inserted into all mother-cell membranes. The protein then achieves proper localization by selective degradation (dashed lines) in the cytoplasmic membrane. Alternatively, randomly inserted SpoIVFB-GFP diffuses and is captured in the engulfing septal membrane. (before engulfment is complete) and then become captured in the fusion protein is initially inserted into all membranes of the the outer forespore membrane. mother cell but is selectively eliminated from the cytoplasmic With SpoIVFB-GFP as a model polytopic membrane protein membrane by proteolytic degradation (Fig. 1B). We have shown and taking advantage of the requirement for SpoIVFA in the that proper localization of SpoIVFB-GFP requires SpoIVFA, localization of SpoIVFB-GFP, we attempted to distinguish which exists in a complex with SpoIVFB-GFP in the engulfing among the three models for how proper localization of an septal membrane (10). In the absence of SpoIVFA, SpoIVFB- integral membrane protein is achieved. The results of our GFP is distributed uniformly in all membranes of the mother cell analysis are most easily compatible with a diffusion-and-capture (Fig. 2, compare A and B). Because SpoIVFA is itself restricted model in which SpoIVFB-GFP is inserted into the cytoplasmic to the engulfing forespore membrane, a model in which membrane from which it diffuses to, and is captured in, the SpoIVFA is responsible for degrading SpoIVFB in the cyto- membranes that surround the forespore during the process of plasmic membrane seems implausible. Conversely, however, engulfment. A further and unexpected finding to emerge from SpoIVFA could be responsible for protecting SpoIVFB-GFP in our investigation is that the outer forespore membrane is ap- the septal membrane from degradation by uniformly distributed parently refractory to membrane protein insertion.

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