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Why, man, I have gotten a lot of results. I know several thousand things that won't work. Thomas A. Edison alkuo.pmd 3 3/7/2005, 1:14 PM CONTENTS LIST OF ORIGINAL PUBLICATIONS ABBREVIATIONS SUMMARY ................................................................................ 1 INTRODUCTION .............................................................................. 2 1. The secretory pathway ............................................................... 2 1.1 Entering the ER..................................................................... 3 1.1.1 Signal peptides ............................................................. 3 1.1.2 Cotranslational translocation ............................................ 3 1.1.3 Posttranslational translocation .......................................... 6 1.2 Modifications in the ER ........................................................... 6 1.2.1 Signal peptide cleavage ................................................... 7 1.2.2 Glycosylation ................................................................ 7 N-Glycosylation ............................................................. 7 O-Glycosylation ............................................................. 9 Glypiation ..................................................................10 1.2.3 Protein folding .............................................................11 Folding enzymes ...........................................................11 Chaperones .................................................................12 1.3 Modifications in the Golgi complex ............................................14 1.3.1 Modifications of N-glycans ...............................................14 1.3.2 Elongation of O-glycans ..................................................15 1.3.3 Precursor processing ......................................................16 2. The molecular mechanisms of vesicular transport between the ER and the Golgi complex ......................................................16 2.1 Anterograde traffic ...............................................................17 2.1.1 COPII proteins ..............................................................17 Sec23p/24p complex ......................................................18 Sec13p/31p complex ......................................................18 Sar1p ........................................................................19 Sec12p .......................................................................19 Sec16p .......................................................................20 2.1.2 Generation of COPII carriers ............................................20 2.1.3 Cargo sorting ...............................................................23 ER exit motifs on cargo proteins .......................................23 Cargo selection by the Sec24 family proteins ........................26 Involvement of Sar1p in cargo selection ..............................27 Other factors involved in sorting .......................................27 2.2 Retrograde traffic.................................................................27 2.2.1 COPI proteins ..............................................................28 2.2.2 Generation of COPI carriers .............................................29 2.2.3 Recruitment of cargo to COPI vesicles ................................30 2.2.4 Role of COPI in anterogradre traffic...................................33 alkuo.pmd 4 3/10/2005, 9:18 AM 2.3 Delivery of cargo to the target organelle .....................................33 2.3.1 Vesicle tethering to target membrane ................................33 2.3.2 Fusion with target membrane ..........................................34 2.3.3 Coordinating directionality of traffic .................................35 AIMS OF THE STUDY .......................................................................37 MATERIALS AND METHODS ...............................................................38 RESULTS AND DISCUSSION ................................................................40 1. Glycosylation of proteins trapped in the yeast ER (I) .........................40 1.1 N-glycans are extended in the ER when ER-to-Golgi traffic is blocked ..40 1.2 Och1p relocates to the ER when COPII-mediated traffic is impaired ....41 1.3 Recycling of Och1p is mediated by COPI ......................................42 1.4 The recycled, but not de novo synthesized Och1p is active in the ER ...42 1.5 O-glycans are extended in the ER when ER-to-Golgi traffic is blocked ..43 1.6 Activity of the recycling mannosyltransferases is only detectable when normal anterograde transport is blocked .............................43 1.7 Biological function of recycling Golgi-enzymes ..............................44 2. Role of the Sec24p family members in ER exit of the secretory glycoprotein Hsp150 (II, III).......................................45 2.1