Abstract
Autophagy is a conserved degradation process in which autophagosomes are generated by cooperative actions of multiple autophagy-related (Atg) proteins. Previous studies using the model yeast Saccharomyces cerevisiae have provided various insights into the molecular basis of autophagy; however, because of the modest stability of several Atg proteins, structural and biochemical studies have been limited to a subset of Atg proteins, preventing us from understanding how multiple Atg proteins function cooperatively in autophagosome formation. With the goal of expanding the scope of autophagy research, we sought to identify a novel organism with stable Atg proteins that would be advantageous for in vitro analyses. Thus, we focused on a newly isolated thermotolerant yeast strain, Kluyveromyces marxianus DMKU3-1042, to utilize as a novel system elucidating autophagy. We developed experimental methods to monitor autophagy in K. marxianus cells, identified the complete set of K. marxianus Atg homologs, and confirmed that each Atg homolog is engaged in autophagosome formation. Biochemical and bioinformatic analyses revealed that recombinant K. marxianus Atg proteins have superior thermostability and solubility as compared with S. cerevisiae Atg proteins, probably due to the shorter primary sequences of KmAtg proteins. Furthermore, bioinformatic analyses showed that more than half of K. marxianus open reading frames are relatively short in length. These features make K. marxianus proteins broadly applicable as tools for structural and biochemical studies, not only in the autophagy field but also in other fields.
Highlights
Autophagosome formation is mediated by multiple autophagy-related (Atg) proteins
We analyzed overall sequence alignments between K. marxianus and S. cerevisiae proteins (3,355 open reading frames (ORFs) pairs from 4,564 K. marxianus ORFs and 5,882 S. cerevisiae ORFs) using the BLAST and ClustalW web servers (Fig. 1A). These analyses showed that housekeeping proteins such as ribosomal proteins, mitochondrial proteins, and proteins involved in metabolism and nutrient utilization are nearly of the same length in S. cerevisiae and K. marxianus (Fig. 1A and supplemental Table 2), potentially due to the fundamental importance of these proteins in cell proliferation
We first identified a complete set of KmAtg proteins essential for autophagosome formation in K. marxianus (Fig. 3), most of which can, at least in part, functionally substitute their counterpart ScAtg proteins in S. cerevisiae (Fig. 5)
Summary
Autophagosome formation is mediated by multiple autophagy-related (Atg) proteins. Autophagy is a conserved degradation process in which autophagosomes are generated by cooperative actions of multiple autophagy-related (Atg) proteins. Previous studies using the model yeast Saccharomyces cerevisiae have provided various insights into the molecular basis of autophagy; because of the modest stability of several Atg proteins, structural and biochemical studies have been limited to a subset of Atg proteins, preventing us from understanding how multiple Atg proteins function cooperatively in autophagosome formation. Ianus open reading frames are relatively short in length These features make K. marxianus proteins broadly applicable as tools for structural and biochemical studies, in the autophagy field and in other fields. Previous studies using the yeast Saccharomyces cerevisiae identified nearly 40 autophagy-related (Atg) proteins involved in various types of autophagy [3, 4, 8]. These Atg proteins are functionally and hierarchically classified into six subgroups as follows: the Atg complex (Atg, Atg, Atg, Atg, and Atg31); a vesicular membrane protein required for the early step of autophagosome formation (Atg9); the autophagy-specific PtdIns 3-kinase complex
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