scholarly journals Apg5p Functions in the Sequestration Step in the Cytoplasm-to-Vacuole Targeting and Macroautophagy Pathways

2000 ◽  
Vol 11 (3) ◽  
pp. 969-982 ◽  
Author(s):  
Michael D. George ◽  
Misuzu Baba ◽  
Sidney V. Scott ◽  
Noboru Mizushima ◽  
Brian S. Garrison ◽  
...  
Keyword(s):  
Biochemical Analysis ◽  
Temperature Sensitive ◽  
Vesicle Formation ◽  
Concerted Action ◽  
Sensitive Mutant ◽  
Temperature Sensitive Mutant ◽  
Dynamic Events

The cytoplasm-to-vacuole targeting (Cvt) pathway and macroautophagy are dynamic events involving the rearrangement of membrane to form a sequestering vesicle in the cytosol, which subsequently delivers its cargo to the vacuole. This process requires the concerted action of various proteins, including Apg5p. Recently, it was shown that another protein required for the import of aminopeptidase I (API) and autophagy, Apg12p, is covalently attached to Apg5p through the action of an E1-like enzyme, Apg7p. We have undertaken an analysis of Apg5p function to gain a better understanding of the role of this novel nonubiquitin conjugation reaction in these import pathways. We have generated the first temperature-sensitive mutant in the Cvt pathway, designated apg5 ts. Biochemical analysis of API import in theapg5 ts strain confirmed that Apg5p is directly required for the import of API via the Cvt pathway. By analyzing the stage of API import that is blocked in theapg5 ts mutant, we have determined that Apg5p is involved in the sequestration step and is required for vesicle formation and/or completion.

scholarly journals Cvt9/Gsa9 Functions in Sequestering Selective Cytosolic Cargo Destined for the Vacuole

2001 ◽  
Vol 153 (2) ◽  
pp. 381-396 ◽  
Author(s):  
John Kim ◽  
Yoshiaki Kamada ◽  
Per E. Stromhaug ◽  
Ju Guan ◽  
Ann Hefner-Gravink ◽  
...  
Keyword(s):  
Temperature Sensitive ◽  
Vesicle Formation ◽  
Sensitive Mutant ◽  
Direct Role ◽  
Vacuole Membrane ◽  
Cytoplasmic Material ◽  
Transport Vesicle ◽  
Membrane Compartment ◽  
Selective Delivery

Three overlapping pathways mediate the transport of cytoplasmic material to the vacuole in Saccharomyces cerevisiae. The cytoplasm to vacuole targeting (Cvt) pathway transports the vacuolar hydrolase, aminopeptidase I (API), whereas pexophagy mediates the delivery of excess peroxisomes for degradation. Both the Cvt and pexophagy pathways are selective processes that specifically recognize their cargo. In contrast, macroautophagy nonselectively transports bulk cytosol to the vacuole for recycling. Most of the import machinery characterized thus far is required for all three modes of transport. However, unique features of each pathway dictate the requirement for additional components that differentiate these pathways from one another, including at the step of specific cargo selection. We have identified Cvt9 and its Pichia pastoris counterpart Gsa9. In S. cerevisiae, Cvt9 is required for the selective delivery of precursor API (prAPI) to the vacuole by the Cvt pathway and the targeted degradation of peroxisomes by pexophagy. In P. pastoris, Gsa9 is required for glucose-induced pexophagy. Significantly, neither Cvt9 nor Gsa9 is required for starvation-induced nonselective transport of bulk cytoplasmic cargo by macroautophagy. The deletion of CVT9 destabilizes the binding of prAPI to the membrane and analysis of a cvt9 temperature-sensitive mutant supports a direct role of Cvt9 in transport vesicle formation. Cvt9 oligomers peripherally associate with a novel, perivacuolar membrane compartment and interact with Apg1, a Ser/Thr kinase essential for both the Cvt pathway and autophagy. In P. pastoris Gsa9 is recruited to concentrated regions on the vacuole membrane that contact peroxisomes in the process of being engulfed by pexophagy. These biochemical and morphological results demonstrate that Cvt9 and the P. pastoris homologue Gsa9 may function at the step of selective cargo sequestration.

scholarly journals Bir1 Is Required for the Tension Checkpoint

2009 ◽  
Vol 20 (3) ◽  
pp. 915-923 ◽  
Author(s):  
Michelle M. Shimogawa ◽  
Per O. Widlund ◽  
Michael Riffle ◽  
Michael Ess ◽  
Trisha N. Davis
Keyword(s):  
Aurora B ◽  
Temperature Sensitive ◽  
Sensitive Mutant ◽  
Checkpoint Response ◽  
Temperature Sensitive Mutant ◽  
The Third ◽  
Chromosomal Passenger ◽  
Chromosomal Passenger Proteins ◽  
Passenger Proteins

The Saccharomyces cerevisiae chromosomal passenger proteins Ipl1 (Aurora B) and Sli15 (INCENP) are required for the tension checkpoint, but the role of the third passenger, Bir1, is controversial. We have isolated a temperature-sensitive mutant (bir1-107) in the essential C-terminal region of Bir1 known to be required for binding to Sli15. This allele reveals a checkpoint function for Bir1. The mutant displays a biorientation defect, a defective checkpoint response to lack of tension, and an inability to detach mutant kinetochores. Ipl1 localizes to aberrant foci when Bir1 localization is disrupted in the bir1-107 mutant. Thus, one checkpoint role of Bir1 is to properly localize Ipl1 and allow detachment of kinetochores. Quantitative analysis indicates that the chromosomal passengers colocalize with kinetochores in G1 but localize between kinetochores that are under tension. Bir1 localization to kinetochores is maintained in an mcd1-1 mutant in the absence of tension. Our results suggest that the establishment of tension removes Ipl1, Bir1, and Sli15, and their kinetochore detachment activity, from the vicinity of kinetochores and allows cells to proceed through the tension checkpoint.

scholarly journals The conserved role of Smu1 in splicing is characterized in its mammalian temperature-sensitive mutant

2006 ◽  
Vol 119 (23) ◽  
pp. 4944-4951 ◽  
Author(s):  
K. Sugaya ◽  
E. Hongo ◽  
Y. Ishihara ◽  
H. Tsuji
Keyword(s):  
Temperature Sensitive ◽  
Sensitive Mutant ◽  
Temperature Sensitive Mutant
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