2. FIG. 1. Schematic representation of the type I, II, III, and IV protein secretion systems. The type I pathway is exemplified by hemolysin A (HlyA) secretion in E. coli, the type III system is exemplified by Yop secretion in Yersinia, the type II system is exemplified by pullulanase secretion in Klebsiella oxytoca, and the type IV system is exemplified by the VirB system in A. tumefaciens. ATP hydrolysis by HlyB, YscN, SecA, and VirB11 is indicated. Secreted effector molecules are depicted as grey ovals. The type II and in some cases the type IV secretion systems utilize the cytoplasmic chaperone SecB, although the Tat export pathway has recently been implicated in the secretion of molecules via the type II pathway. Type III secretion also involves cytoplasmic chaperones (SycE); however, they do not interact with the Sec inner membrane translocon. The major structural proteins of each system are depicted in relation to their known or deduced position in the cell envelope. EM, extracellular milieu; OM, outer membrane; Peri, periplasm; IM, inner membrane; Cyto, cytoplasm.

3. Homework: Analyze the following protein sequences and decide if these proteins have a predicted signal sequence. If a protein DOES have a signal sequence, what does this mean? Where would you expect to find the protein? The cytoplasm? The periplasm? The culture medium? What if a protein DOES NOT have a signal sequence? Where would you expect to find this protein?

4. MNNNDLFQASRRRFLAQLGGLTVAGMLGPSLLTPRRATAAQAATDAVISKEGILTGSHWG AIRATVKDGRFVAAKPFELDKYPSKMIAGLPDHVHNAARIRYPMVRVDWLRKRHLSDTSQ RGDNRFVRVSWDEALDMFYEELERVQKTHGPSALLTASGWQSTGMFHNASGMLAKAIALH GNSVGTGGDYSTGAAQVILPRVVGSMEVYEQQTSWPLVLQNSKTIVLWGSDLLKNQQANW WCPDHDVYEYYAQLKAKVAAGEIEVISIDPVVTSTHEYLGREHVKHIAVNPQTDVPLQLA LAHTLYSENLYDKNFLANYCVGFEQFLPYLLGEKDGQPKDAAWAEKLTGIDAETIRGLAR QMAANRTQIIAGWCVQRMQHGEQWAWMIVVLAAMLGQIGLPGGGFGFGWHYNGAGTPGRK GVILSGFSGSTSIPPVHDNSDYKGYSSTIPIARFIDAILEPGKVINWNGKSVKLPPLKMC IFAGTNPFHRHQQINRIIEGLRKLETVIAIDNQWTSTCRFADIVLPATTQFERNDLDQYG NHSNRGIIAMKQVVPPQFEARNDFDIFRELCRRFNREEAFTEGLDEMGWLKRIWQEGVQQ GKGRGVHLPAFDDFWNNKEYVEFDHPQMFVRHQAFREDPDLEPLGTPSGLIEIYSKTIAD MNYDDCQGHPMWFEKIERSHGGPGSQKYPLHLQSVHPDFRLHSQLCESETLRQQYTVAGK EPVFINPQDASARGIRNGDVVRVFNARGQVLAGAVVSDRYAPGVARIHEGAWYDPDKGGE PGALCKYGNPNVLTIDIGTSQLAQATSAHTTLVEIEKYNGTVEQVTAFNGPVEMVAQCEY VPASQVKS

5. MKIKTGARILALSALTTMMFSASALAKIEEGKLVIWINGDKGYNGLAEVGKKFEKDTGIK VTVEHPDKLEEKFPQVAATGDGPDIIFWAHDRFGGYAQSGLLAEITPDKAFQDKLYPFTW DAVRYNGKLIAYPIAVEALSLIYNKDLLPNPPKTWEEIPALDKELKAKGKSALMFNLQEP YFTWPLIAADGGYAFKYENGKYDIKDVGVDNAGAKAGLTFLVDLIKNKHMNADTDYSIAE AAFNKGETAMTINGPWAWSNIDTSKVNYGVTVLPTFKGQPSKPFVGVLSAGINAASPNKE LAKEFLENYLLTDEGLEAVNKDKPLGAVALKSYEEELAKDPRIAATMENAQKGEIMPNIP QMSAFWYAVRTAVINAASGRQTVDEALKDAQTRITK

6. MAKQPGLDFQSAKGGLGELKRRLLFVIGALIVFRIGSFIPIPGIDAAVLAKLLEQQRGTI IEMFNMFSGGALSRASIFALGIMPYISASIIIQLLTVVHPTLAEIKKEGESGRRKISQYT RYGTLVLAIFQSIGIATGLPNMPGMQGLVINPGFAFYFTAVVSLVTGTMFLMWLGEQITE RGIGNGISIIIFAGIVAGLPPAIAHTIEQARQGDLHFLVLLLVAVLVFAVTFFVVFVERG QRRIVVNYAKRQQGRRVYAAQSTHLPLKVNMAGVIPAIFASSIILFPATIASWFGGGTGW NWLTTISLYLQPGQPLYVLLYASAIIFFCFFYTALVFNPRETADNLKKSGAFVPGIRPGE QTAKYIDKVMTRLTLVGALYITFICLIPEFMRDAMKVPFYFGGTSLLIVVVVIMDFMAQV QTLMMSSQYESALKKANLKGYGR

7. To analyze these proteins sequences, go to http://www.cbs.dtu.dk/services/SignalP/. Once at this site, copy and paste the sequence into the submission box. Under the “Organism group” heading, select gram-negative bacteria as these sequences come from E. coli. Then click “Submit”. The next page will be the prediction results. This will tell you whether or not the proteins have a predicted signal sequence. http://www.cbs.dtu.dk/services/SignalP/ I will do a demo in class Monday.