Assignment for subcellular protein localization 1. 	Name of chosen protein:	HWP1   	Organism: Candida albicans 	Locus: CAU64206 	NCBI Nucleotide accession #: U64206 	NCBI protein accession #: AAC96368 2.	Predicted function: HWP1 possibly functions as an adhesin in Candida albicans. It has also been demonstrated to play a role in the morphological change (from yeast form to hyphae form) of Candida albicans. 3.	The adhesin function of HWP1 implies that it localizes to the cell wall. I did not find any data directly proving this though. In addition, I think it would be interesting to check the change of HWP1 localization during the morphological change of Candida albicans.  4. Results from different protein localization: I. TargetP 1.1 Server - prediction results (Technical University of Denmark) TargetP 1.1 predicts the subcellular location of eukaryotic proteins. The location assignment is based on the predicted presence of any of the N-terminal presequences: chloroplast transit peptide (cTP), mitochondrial targeting peptide (mTP) or secretory pathway signal peptide (SP). ### targetp v1.1 prediction results ################################## Cleavage site predictions not included. Using NON-PLANT networks. In a short word, TargetP 1.1 predicts HWP1 will go through the secretory pathway, and it contains a secretory pathway signal peptide (SP).  Final Results:  48.0 %: extracellular, including cell wall 12.0 %: cytoplasmic 12.0 %: nuclear 12.0 %: endoplasmic reticulum 8.0 %: vesicles of secretory system 4.0 %: plasma membrane 4.0 %: mitochondrial >> prediction for queryProtein is exc WOLFPSORT is an updated version of PSORTII. Comparing the results from two programs, we can see that they gave same/similar results in most aspects. Both programs said that HWP1 doesn't seem to have an N-terminal signal peptide. HWP1 looks more like a peripheral protein rather than an integral one, with single trans-membrane sequence near its C-terminus and its N-terminus facing inside the cell. Nonetheless, those two programs gave controversial results of HWP1 localization. PSORTII suggests that HWP1 localizes to nucleus, while WOLFPSORT predicts HWP1 localizes to extracellular space or cell wall. Given adhesion function of HWP1, the result of WOLFPSORT makes more sense. And it is consistent with the result of TargetP (Because WOLFPSORT has been updated?) However, we can not rule out the possibility that HWP1 may localize to nucleus.  5. HWP1 were overall predicted to be a peripheral membrane protein by all the programs. I further used iPSORT to do an AAindex Analysis, and got Based on this Hydropathy Index of HWP1, I would predict a trans-membrane sequence at its N-terminus. (If I have understood the hydropathy index correctly) I don't know why both PSORTII and WOLFPSORT predict a trans-membrane sequence at the C-terminus of HWP1. 6. Although either PSORTII or WOLFPSORT did not find any N-terminal signal peptide, iPSORT does find a signal peptide in the N-terminus of HWP1. This is consistent with and further consolidates the result of TargetP, which says HWP1 has a signal sequence and is likely a secretory protein. 7. Base on the results of prediction programs, as well as previous data in literature. I would propose that HWP1 has a low expression level in the yeast-form cells of Candida. The expression of HWP1 will be induced during Candida cells change from yeast-form to hyphae-form, and HWP1 will localize to the cell wall. 8. Considering the expression level may affect a protein's subcellular localization, I would take the advantage of homologous recombination in Candida, and replace the endogenous gene of HWP1 with a fusion gene in which HWP1 being tagged with a fluorophore. In this case, the fusion gene will be expressed under endogenous promoter of HWP1, and hopefully has a similar expression level as HWP1. Some better-known adhesion (e.g. ALS3) will be used as cell wall marker. I expected to see dim or no fluorescence from fusion protein in yeast-form cells. After shifting conditions and cells becoming hyphae, I expect to see strong signal and the fluorescence localizes to the cells' surface. If I also tagged ALS3 with another fluorophore, I expect to see co-localization of HWP1 and ALS3. A possible problem is the fluorophore may affect the localization of HWP1. An alternative way to get around is to use HWP1 antibody (which we already have) to do immuno-staining. The down-side of this approach is non-specific binding and staining.  Both approaches may require certain amount of HWP1 to exist inside cells. If HWP1 keeps being expressed at a low level, we can make Candida cultures, do cell fractionation. Use HWP1 antibody to detect the amount of HWP1 in each fraction. Candida albicans hyphal wall protein 1 (HWP1) gene, complete cds The Michigan Corpus of Upper-level Student Papers (MICUSP) is owned by the Regents of the University of Michigan (UM), who hold the copyright. The corpus has been developed by researchers at the UM English Language Institute. The corpus files are freely available for study, research and teaching. However, if any portion of this material is to be used for commercial purposes, such as for textbooks or tests, permission must be obtained in advance and a license fee may be required. For further information about copyright permissions, please contact micusp-help@umich.edu. The recommended citation for MICUSP is: Michigan Corpus of Upper-level Student Papers. (2009). Ann Arbor, MI: The Regents of the University of Michigan.