![[Feedback]](/icons/banner/feedbackACT.gif){kind=icon}
![[For Subscribers]](/icons/banner/subscriberACT.gif){kind=icon}
![[Archive]](/icons/banner/archiveACT.gif){kind=icon}
![[Search]](/icons/banner/searchACT.gif){kind=icon}
![[Contents]](/icons/banner/tocACT.gif){kind=icon}
|
|
|
|
|
||
Vol. 9, Issue 4, 817-827, April 1998
-Lactamase in the Yeast Cytoplasm before
Translocation into the Endoplasmic Reticulum
Institute of Biotechnology, University of Helsinki, 00014 Helsinki,
Finland
Polypeptides targeted to the yeast endoplasmic reticulum (ER)
posttranslationally are thought to be kept in the cytoplasm in an
unfolded state by Hsp70 chaperones before translocation. We show here
that Escherichia coli 
-lactamase associated with Hsp70, but adopted a native-like conformation before translocation in
living Saccharomyces cerevisiae cells. -Lactamase is
a globular trypsin-resistant molecule in authentic form. For these
studies, it was linked to the C terminus of a yeast polypeptide
Hsp150
, which conferred posttranslational translocation and provided
sites for O-glycosylation. We devised conditions to
retard translocation of Hsp150--lactamase. This enabled us to
show by protease protection assays that an unglycosylated precursor was
associated with the cytoplasmic surface of isolated microsomes, whereas
a glycosylated form resided inside the vesicles. Both proteins were
trypsin resistant and had similar -lactamase activity and
Km values for nitrocefin. The enzymatically
active cytoplasmic intermediate could be chased into the ER, followed
by secretion of the activity to the medium. Productive folding in the
cytoplasm occurred in the absence of disulfide formation, whereas in
the ER lumen, proper folding required oxidation of the sulfhydryls.
This suggests that the polypeptide was refolded in the ER and
consequently, at least partially unfolded for translocation.
This article has been cited by other articles:
|
|
 |
|
  L. Seppa and M. Makarow Regulation and Recovery of Functions of Saccharomyces cerevisiae Chaperone BiP/Kar2p after Thermal Insult Eukaryot. Cell, December 1, 2005; 4(12): 2008 - 2016. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 N. Fatal, L. Karhinen, E. Jokitalo, and M. Makarow Active and specific recruitment of a soluble cargo protein for endoplasmic reticulum exit in the absence of functional COPII component Sec24p J. Cell Sci., May 1, 2004; 117(9): 1665 - 1673. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
L. Karhinen and M. Makarow Activity of recycling Golgi mannosyltransferases in the yeast endoplasmic reticulum J. Cell Sci., January 15, 2004; 117(2): 351 - 358. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. Yabal, S. Brambillasca, P. Soffientini, E. Pedrazzini, N. Borgese, and M. Makarow Translocation of the C Terminus of a Tail-anchored Protein across the Endoplasmic Reticulum Membrane in Yeast Mutants Defective in Signal Peptide-driven Translocation J. Biol. Chem., January 24, 2003; 278(5): 3489 - 3496. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 N. Fatal, T. Suntio, and M. Makarow Selective Protein Exit from Yeast Endoplasmic Reticulum in Absence of Functional COPII Coat Component Sec13p Mol. Biol. Cell, December 1, 2002; 13(12): 4130 - 4140. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. Geissler, J. Rassow, N. Pfanner, and W. Voos Mitochondrial Import Driving Forces: Enhanced Trapping by Matrix Hsp70 Stimulates Translocation and Reduces the Membrane Potential Dependence of Loosely Folded Preproteins Mol. Cell. Biol., October 15, 2001; 21(20): 7097 - 7104. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A.-L. Hänninen, M. Simola, N. Saris, and M. Makarow The Cytoplasmic Chaperone Hsp104 Is Required for Conformational Repair of Heat-denatured Proteins in the Yeast Endoplasmic Reticulum Mol. Biol. Cell, November 1, 1999; 10(11): 3623 - 3632. [Abstract] [Full Text]
|
|
|
|
|
|
J. L. Brodsky, E. D. Werner, M. E. Dubas, J. L. Goeckeler, K. B. Kruse, and A. A. McCracken The Requirement for Molecular Chaperones during Endoplasmic Reticulum-associated Protein Degradation Demonstrates That Protein Export and Import Are Mechanistically Distinct J. Biol. Chem., February 5, 1999; 274(6): 3453 - 3460. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
T Suntio, A Shmelev, M Lund, and M Makarow The sorting determinant guiding Hsp150 to the COPI-independent transport pathway in yeast J. Cell Sci., January 11, 1999; 112(22): 3889 - 3898. [Abstract] [PDF]
|
|
|
|
|
|
E. Paunola, M. Qiao, A. Shmelev, and M. Makarow Inhibition of Translocation of beta -Lactamase into the Yeast Endoplasmic Reticulum by Covalently Bound Benzylpenicillin J. Biol. Chem., September 7, 2001; 276(37): 34553 - 34559. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. Palmgren, P. J. Ojala, M. A. Wear, J. A. Cooper, and P. Lappalainen Interactions with PIP2, ADP-actin monomers, and capping protein regulate the activity and localization of yeast twinfilin J. Cell Biol., October 15, 2001; 155(2): 251 - 260. [Abstract] [Full Text] [PDF]
|
|
